Fig. 3. Identification of individual brain NBs at different embryonic stages. (A) Deadpan (Dpn) expression in the procephalic neuroectoderm by mid stage 8 (mst8) at two different focal planes (flat preparation; anterior is towards top, dorsal is leftwards). Dpn protein is found in two small spots in the ocular (arrowheads in A), and in a stripe-like domain in the antennal ectoderm (black arrows) as well as in the first brain NBs (Pcv7, 9), in a focal plane about 10 µm deeper (A'). Red arrows indicate same positions in the two optic foci, revealing that Pcv7 derives from a small cluster of Dpn-positive ectodermal cells. (B-H) svp-lacZ (brown)/Engrailed (blue) double stainings in flat preparations (B,D,F,H; ventral views, anterior (a) is towards the top and dorsal (d) is leftwards) and wholemounts (C,E,G; lateral views, anterior is towards the top and dorsal is towards the left). Individual NBs are identifiable by marker expression, morphology (size), and position relative to each other. Compare with semi-schematic presentation in Fig. 2. (B) Early stage 9 (est9); svp is weakly expressed in a subset of NBs. Ectodermal progenitor cells immediately prior to NB formation are marked by brackets (untypically, in this embryo Dv4 develops before Dd7). (CH) For comparison, whole-mount and flat preparation of the same individuals are shown. (C,D) Late stage 9 (lst9). Note the higher optic resolution and easier identification of NBs in the flat preparation. (E,F) Mid stage 10 (mst10; F, the most dorsal Svp-positive Pcd18 was removed accidentally during preparation). (G,H) Late stage 11 (lst11). a, d: anterior, dorsal; as, is: antennal and intercalary en stripe, respectively; cl: en expression in the clypeolabrum; hs: en head spot; CL: clypeolabrum; FG: foregut.

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