Fig. S6

Reelin gradient is required for VLDLR-mediated chemoattraction of retinal afferents during laminar targeting. Related to Figure 6

(A) Side-view of the retinotectal projection of a 5 dpf reln ^-/- larva expressing the Tg(isl2b:Gal4;UAS:RFP) transgene that labels the entire RGC population (magenta). Membrane-targeted lyn-GFP is transiently expressed as a control in a mosaic subset of these RGCs. D = dorsal; A = anterior. Scale bar = 35 μm.

(B) Side-view of the retinotectal projection showing the entire RGC population (magenta) in 5 dpf reln ^-/- larva expressing Tg(isl2b:Gal4;UAS:RFP). In addition vldlr-GFP is transiently expressed in a mosaic subset of RGCs. In the absence of reelin expression VLDLR overexpressing cells are able to target the deeper SGC and SAC layers with the same frequency than control cells. D = dorsal; A = anterior. Scale bar = 35 μm.

(C) Quantification (see A and B) showing the percentage of larvae displaying one or more GFP-labeled RGC axons in the main layers of the tectal neuropil (n=16 UAS:lyn-GFP expressing larvae; n=16 UAS:vldlr-GFP expressing larvae). The superficial layers SO and SFGS were merged and scored together, as their close proximity makes their discrimination in isl2b:Gal4 larvae difficult. In addition, because of the targeting mistakes seen in 5 dpf reln ^-/- larva RGCs, SGC and SAC layers were also merged and scored together as their distinction was uncertain in this mutant background. RCGs overexpressing vldlr-GFP target the deep neuropil layers at a similar frequency compared to RGCs expressing lyn-GFP (ctrl) suggesting in the absence of Reelin, VLDLR has no effect on layer preference. SO, stratum opticum; SFGS, stratum fibrosum et griseum superficiale; SGC, stratum griseum centrale; SAC, stratum album centrale; SPV, stratum periventriculare.

(D) Quantification (see A and B) of RGC projection thickness. Since layer assignment is more difficult in 5 dpf reln ^-/- larvae, we scored the phenotype as the total thickness of the projection as measured by taking the distance between the most superficial and the deepest visible axonal branch. The average thickness of the RGC projection in reln ^-/- larvae was 38.0 μm ± 2.8 μm s.e.m (n=16 larvae) for UAS:lynGFP expressing RGCs and 40.6 μm ± 2.9 μm s.e.m (n=16 larvae) for UAS:vldlr-GFP expressing RGCs.