Fig. 5. Dorsal progression of ZLI differentiation requires continuous Shh signaling. (A) Strategy to test the requirement for Shh signaling during the 48-hour explant culture period. 25 ng/µl MAb-5E1 was added to intact stage 13-14 forebrain explants after a defined period in culture, and the relative length of the ZLI was assessed after 48 hours. (B-G) Addition of MAb-5E1 from the time of explant isolation (C) drastically reduced the length of the ZLI compared with untreated controls (B). MAb-5E1 treatment of stage 11 forebrain explants completely eliminated Shh expression from the ZLI (D). MAb-5E1 addition after 12 (E), 18 (F) and 24 (G) hours in culture arrested the dorsal progression of ZLI differentiation. (H) MAb-5E1 addition at any point during the first 28 hours of the culture period prevented the dorsal progression of the ZLI (blue line) compared with treatment with control serum fixed after corresponding periods in culture (red line). Error bars represent s.e.m.