Fig. 1. Characterization of the superficial migration in vivo and in vitro. Transverse sections of E12.5 (A-C) and E13.5 (D-F) mouse hindbrain at the level of the superficial migration. Cresyl Violet staining (A,D), in situ hybridization (B,E) and immunocytochemistry (C,F) for TAG-1. At E12.5 the cells of the superficial stream have not started their migration yet (A) but they have just been generated at the rhombic lip (asterisk in B). TAG-1 protein is widely expressed at the superficially located fibers (arrows in C). At E13.5, the migrating TAG-1-positive cells have reached and crossed the midline (arrowheads in D-F). (G) Schematic representation of the dissection procedure. The hindbrains of E12.5 embryos were dissected, cut at the dorsal midline and cultured as an ‘open book’ configuration on Biopore membranes for the appropriate time (days in vitro, DIV). The region of the migration is marked in red and the arrows indicate the direction of the migration on the explant. (H) Transverse section of an explant taken at E12.5 at the region of the superficial migration. BrdU and TAG-1 staining is distinct. At the region of the rhombic lip mitotic (BrdU positive) cells are detected at the most lateral side and TAG-1 transcripts are expressed only by those postmitotic cells that have started to migrate ventrally (arrow). BrdU-positive cells are also detected in the ventricular zone while TAG-1 signal is found in the subventricular zone. (I) Diagram of a normal explant on E12.5 at the beginning of culture period. Arrows indicate the direction of the migration within the explant. (J-L) Whole-mount in situ hybridization for TAG-1 on explants at E12.5 (J), E12.5 + 1DIV (K) and E12.5 + 2 DIV (L). At E12.5, the cells of the superficial stream have just been generated at the rhombic lip (asterisk in J). One day later they have started to migrate (between the broken lines in K) but have not reached the midline yet, while 2 days later they have crossed the midline (L). The TAG-1-positive cells form characteristic stripes as they migrate. (M-N) Diagram of an explant with a unilateral rhombic lip ablation (M) and whole-mount in situ hybridization for TAG-1 on ablated explant after 2 DIV (N). TAG-1-positive cells have crossed the midline and they still express TAG-1 transcripts. (O,P) Confocal images of whole-mount immunocytochemistry for TAG-1 on ablated explants. TAG-1 protein is expressed by the cells on the normal (left) side of the explant (O), but it is also expressed by the migrating cells after crossing the midline, at the ablated (right) side of the explant (P). Asterisks indicate the TAG-1-positive cell bodies on the ablated side (P). CB; cerebellum; fbm, facial branchiomotor neurons; FP, floor plate; h, hypoglossal nucleus; RL, rhombic lip; SVZ and s, subventricular zone. Scale bars: 500 µm in A-F,J-N; 200 µm in H; 50 µm in O,P.