Supplemental Experimental Procedures

In situ hybridization

Probe preparation. The in situ hybridization protocol was modified from [46]. Digoxigenin-labeled RNA probes were generated using the DIG RNA labeling kit (SP6/T7) from Roche Biochemicals using the manufacturer’s recommendations. The Sema-5c cDNA clone RE68041 was digested with EcoRV to make sense probes with T7 RNA polymerase and with NotI to make antisense probes with T3 RNA polymerase. Labeled probe was quantified using DIG quantification test strips (Roche Biochemicals).

Tissue fixation and treatment. Brains of larvae and adults were dissected in PBS and stored in PBS on ice for no more than 1 hour. The tissues were fixed for 1 h in 4% formaldehyde in PBS. After 3x 10 min rinsing with PBT, the tissue was digested with proteinase K (15µg/ml) in PBT for 2 min at 37 oC followed by 2 washes of 2 min with glycine (2mg/ml). After extensive washing, tissues were prehybridized for 2 hours in hybridization solution at 60oC. Denatured probe (boiling for 5 min) was added at a final concentration of 0.25 ng/ml and hybridization continued overnight at 60oC. After removal of the probe, the tissue was washed at 60oC (hybridization solution, 30 min; 50% hybridization solution/50% PBT, 30 min; PBT, 30 min), at 37oC (PBT, 2x 30 min), and at room temperature (PBT, 30 min). The tissue was then blocked for 2 hours at room temperature in 1x blocking reagent (Roche Biochemicals) in PBT, followed by incubation with alkaline phosphatase coupled anti-digoxigenin antibody (1:2000) for 1h at room temperature. Following washes (4x 15 min in PBT), the tissue was incubated in staining buffer (0.1M Tris-HCl pH 9.5, 0.1M NaCl) for 20 min. Staining was started by the addition of 5µl/ml NBT (Roche Biochemicals) and 3.75 µl/ml BCIP (Roche Biochemicals) and monitored under a Leica MZ6 dissecting stereomicroscope at regular time intervals until staining was complete. Following dehydration through increasing glycerol concentrations (30% 50% 70% with PBS), brains were mounted on microscope slides.

Wholemountimmunohistochemistry

For larval brains, the anterior part of larvae was inverted for staining and larval brains were further dissected after staining. Staining is as described in the Experimental Procedures. The rabbit polyclonal anti-GFP antibody (AbCam, Cambridge, UK) was used at a dilution of 1:300. Anti-GFP immunohistochemistry was analyzed on a Leica DMRXA epifluorescence microscope.

Supplemental Figure Legend

Supplemental Figure 1. Expression analysis of Sema-5c in the larval brain.

(A) In situ hybridization with digoxigenin-labeled antisense probe on third instar larval brain reveals staining in the optic lobes, in the thoracic ganglion, in the midline glia and in dispersed cells in the brain.

(B) Control in situ hybridization with digoxigenin-labeled sense probe on third instar larval brain shows no staining.

(C-F) Higher magnifications of (A) showing expression of Sema-5c in the outer proliferation center of the optic lobes (C), distinct cells at the base of the brain hemispheres (arrow in D), diffuse staining above background primarily in the thoracic ganglion (E), staining in the midline glia (arrow in F).

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