Fig. 4. Boundary formation requires Hedgehog and Ci. (A-H) Stage 13+ embryos stained with anti-Engrailed (black or brown). (A,B) Wild-type embryos. Deep grooves are easily seen (black arrows) in the lateral view in A, while the ventral view shows the normal stripes of Engrailed expression (two to three cell diameters wide). (C,D) Embryos lacking hedgehog but continuing to express engrailed (full genotype is shown). No groove form as seen from the lateral view focused on the ventral midline (C) and stripes of engrailed-expressing cells are broken up into clumps as seen in the ventral view (D). (E,F) In ci⁹⁴ embryos (with artificially maintained Engrailed), grooves form (black arrows in E) and Engrailed stripes appear normal (F). (G,H) In ci^Cell embryos (with artificially maintained Engrailed), grooves do not form (G) and there is moderate disruption of the Engrailed stripes (H). (I-L) Cartoons summarising the results shown in panels above. (M,N) SEM of stage 13 embryos. Compare the wild type in M with a hedgehog mutant (with artificially maintained Engrailed) in N.