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A combination of chain and neurophilic migration involving the adhesion molecule TAG-1 in the caudal medulla

Katerina Kyriakopoulou¹, Isabel de Diego^2,*, Marion Wassef² and Domna Karagogeos^1,

¹ Department of Basic Science, Medical School, University of Crete and Institute of Molecular Biology and Biotechnology, PO Box 1527, 711 10 Heraklion, Greece
² CNRS UMR 8542, Regionalisation Nerveuse, Ecole Normale Superieure, 46 rue d’Ulm 75230 Paris Cedex 5, France
^* Present address: Departamento de Biología Celular, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos, 29071 Málaga, Spain

View larger version (55K): [in a new window]   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.

View larger version (105K): [in a new window]   Fig. 2. Migrating cells extend long leading processes towards the direction of the migration. Confocal images of whole mount explants injected with DiI at the rhombic lip. The pictures on the right illustrate the regions boxed on the diagrams. (A,B) Explant injected at E12.5. Close to the pial surface long leading processes, labeled with DiI, extended towards the midline (A). At a deeper level, many labeled axons have crossed the midline and turned anteriorly (B). (C,D) Explant injected at E12.5 and fixed after 1DIV. DiI-labeled cells emerged from the rhombic lip. These migrating cells have a unipolar shape with a long leading process. (E) Explant injected at E12.5 and fixed after 2DIV. The migrating cells have already reached the side contralateral to the injection. Scale bars: 50 µm.

View larger version (131K): [in a new window]   Fig. 3. Two different patterns of migration followed by LRN/ECN neurons. Confocal images of whole-mount immunocytochemistry for TAG-1 and Tuj-1 on explants. The pictures on the right illustrate the regions boxed on the diagrams. (A-C) Explants fixed on E12.5. TAG-1 is expressed by superficial transverse fibers (A,C) that are not Tuj-1 positive (B,C). These fibers could coincide with the leading processes of neurons of the superficial migration that are still located at the rhombic lip. (D-F) Explants fixed on E12.5+1DIV. TAG-1 is expressed by the migrating cells emerged by the rhombic lip (D,F). These cells migrate in close contact to each other in an environment void of Tuj-1-positive neuronal fibers (E). The Tuj-1 staining is detected only at the lateral and deeper part of the explant (E,F). (G-L) Explants fixed on E12.5+2DIV. TAG-1-positive cells (G,I) migrating perpendicular to the Tuj-1 positive axonal fibers (H, asterisks in I) are still detected at the lateral part of the explant. Closer to the midline (J-L), TAG-1-positive cells migrate in close contact with neuronal fibers which are Tuj-1 but not TAG-1 positive (arrowheads in L). Scale bars: 50 µm in A-F; 20 µm in G-L.

View larger version (62K): [in a new window]   Fig. 4. Blocking TAG-1 function in vitro alters the migratory behavior of LRN and ECN cells. Whole-mount in situ hybridization for TAG-1 on E12.5 explants after 2-4 days in culture. (A,C,E,G) Blocking the function of TAG-1 by cutting the GPI link with PI-PLC (A), blocking with Fab fragments of polyclonal antibodies against TAG-1 (C), or incubating with control non-depleted medium (E) or medium with soluble TAG-1-Fc (G). (B,D,F,H) Explants incubated in control medium (B), medium with control Fab fragments (D), in antibody-depleted medium (F) and in medium with control soluble protein MUC18-Fc (H). (I,J) Explants incubated in control medium for 2 days and then for 2 more days in medium with polyclonal antibody against TAG-1 (I) or with PI-PLC (J). The migration of TAG-1-positive cells occurred normally. Scale bars: 500 µm.

View larger version (99K): [in a new window]   Fig. 5. The role of L1 and PSA-NCAM in the superficial migration. (A) Staining with antibody against L1. Only some axons at the region of the superficial migration are positive for L1 (arrowheads). (B) Whole-mount in situ hybridization for TAG-1 on explants incubated for 2 days in medium with Fab fragments of polyclonal antibody against L1. The trajectories of the migrating cells are normal. (C,D) Whole-mount in situ hybridization for TAG-1 on explants from L1-null (C) and wild-type (D) animals. The migration of TAG-1-positive cells is normal even in L1-deficient animals. (E) Staining with antibody against PSA-NCAM. PSA-NCAM was detected on the cell bodies of the migrating cells. (F) Whole mount is situ hybridization for TAG-1 on explants treated with EndoN during the culture. The TAG-1-positive cells migrated normally when PSA was cut. Scale bars: 50 µm in A,E; 500 µm in B-D,F.

View larger version (26K): [in a new window]   Fig. 6. Superficial migration. Laterally, the cells migrate in contact with each other, extending long leading processes. Medially, close to the floor plate (lines in black), the cells migrate by following pre-existing axonal fibers. Migrating cells and processes are shown in green, while the tangentially oriented axons are indicated in red.