Fig. 7.

Dscam is necessary to establish BC4 axon tiling. (A–C) Colocalization of DSCAM protein with BC4 axons. All analyses were performed in serial z-stack images; shown here is a single confocal plane from an analyzed stack. (A and B) Cryo-sections of retina (3 mo) stained with anti-DSCAM and HTR:GFP to determine colocalization. (Scale bar, 5 μm.) (A) Image showing DSCAM/BC4 axon colocalization. (B) Axon channel flipped about the horizontal axis as a control for incidental colocalization. Arrowheads point to colocalized DSCAM puncta on BC4 axons. (C) Quantification of DSCAM puncta colocalization per BC4 axon. A significant increase in DSCAM puncta was detected when comparing the original images to the flipped controls (t test, P < 0.001). (D and E) Whole-mount retina (3 mo), BC4s are labeled genetically (HTR:Cre × Ai9 and HTR:GFP or HTR:GFP only). (Scale bar, 20 μm.) (D) Dscam^+/+, BC4 axons outlined in white to visualize tiling. (E) Dscam^{FF HTR:Cre}, axons outlined in white. Only the green axon is Dscam^flox. The others outlined are DscamΔ^flox. Axons invade each other’s territory when one or both lack DSCAM. (F and G) Quantifications of axon area (F) and total overlap with neighboring BC4 axons (G). Dscam^flox BC4 axons become significantly larger (t test < 0.0001) and overlap significantly more with neighboring DscamΔ^flox BC4 axons (t test, P = <0.0001) compared with Dscam^+/+ BC4 axons. *P < 0.05. Sample size WT, n = 30; Δ^flox, n = 30. Error bars = SEM.