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Fig. 3 HS Is Required for Correcting Missorted DN Axons in the Tract(A–B2) DN (A and B, blue in A2 and B2) and VN (red in A′ and B′) retinal axons were topographically labeled at 48 and 72 hpf in dak mutants lacking HS. As observed in WT (Figure 1), some missorted DN axons are observed along VN axons at 48 hpf (arrows). DN (yellow arrowheads) and VN (white arrowhead) growth cones navigate along mixed trajectories (A′). In contrast to WT, however, missorted DN axons remain clearly visible at 72 hpf (arrows). Lateral views, confocal maximal projections; scale bar represents 20 μm. (C and D) Measurement of DiD and DiI signal intensities across the tract shows that missorted DN axons remain from 48 to 72 hpf in dak. (E) Missorting index (MI) is similar at 48, 54, and 72 hpf in dak, showing that missorted DN axons are not corrected. Error bars correspond to SEs. (F–H) Restoring HS synthesis at late stages in dak mutants corrects missorted DN axons. (F and G) Tg(hsp70l:ext2-2A-TagRFP); dak mutant embryos were heat shocked at 39°C for 1 hr at 48 hpf, fixed at 4 dpf, and their DN axons were topographically labeled with DiO. dak mutants carrying the rescue ext2 transgene (Tg+, F) do not show any missorted DN axons, whereas dak sibling mutants lacking the transgene (Tg, G) do (arrows). Lateral views, confocal maximal projections; scale bar represents 20 μm. (H) The proportion of dak mutants with missorted DN axons was scored at 4 dpf after embryos were heat shocked at various stages. In absence of heat shock, around 80% of dak mutants exhibit missorted DN axons. Heat shocks as late as 64 hpf correct missorting of DN axons in Tg+ but not in Tg dak mutants. Error bars correspond to SEs.