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Multiple influences on the migration of precerebellar neurons in the caudal medulla

I. de Diego^1,*,, K. Kyriakopoulou^1,2,, D. Karagogeos² and M. Wassef^1,

¹ CNRS UMR C8542, Régionalisation Nerveuse, niveau 8, Ecole Normale Supérieure 46, rue d’Ulm 75230 Paris Cedex 05, France
² University of Crete Medical School and Institute of Molecular Biology and Biotechnology, PO Box 1527, Heraklion 711 10, Crete, Greece
^* Present address: Departamento de Biología Celular, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos, 29071 Málaga, España
These authors contributed equally to this work

View larger version (88K): [in a new window]   Fig. 1. The superficial and olivary migrations. (A) Schematic representation of a transverse section through the caudal hindbrain. The olivary migrations (OM) gives rise to the ipsilateral inferior olive (ION), whereas the neurons of the superficial migration (SM) form the contralateral lateral reticular (LRN) and external cuneatus (ECN) nuclei. (B-D) Transverse sections of the caudal hindbrain of E13.5 embryos. (B) A gap separates the superficial (arrow) and olivary (ION) migrations on sections double labeled for TAG-1 (brown) and Brn3.2 (red) transcripts. (C) Underlying the olivary migration is a superficial sheet of axons that express and embryonic form of NCAM (e-ncam). (D) Vimentin antibody labels the specialized radial glia that constitutes the floor plate. This tight bundle loosens when it reaches the pial surface, delimiting a superficial path (arrow). (E-J) Transverse sections of the caudal hindbrain treated for the detection of TAG-1 (E-G) or Brn3.2 (H-J). (E-G) Between E11.5 and E14.5, TAG-1 marks the superficial migration (arrows), the hypoglossal nucleus (H) and the subventricular zone (asterisks in E,F). The neurons of the superficial migration reach the midline at E13.5 (F). By E15.5 (G), TAG-1 expression is downregulated. (H-J) Brn3.2 is expressed in the neurons of the olivary migration (arrows) beginning from E12.5 (H), and more faintly in other neuronal populations of the dorsal hindbrain. Notice that the stream of Brn3.2 neurons seems to originate in a gap in the subventricular expression of TAG-1. The olivary neurons reach the midline at E13.5 (I). Brn3.2 expression in the ION persists at later stages (J). Bar, 300 µm.

View larger version (88K): [in a new window]   Fig. 2. The superficial and olivary migrations in vitro. (A-C) BrdU immunostained bulbar explants viewed from the pial surface. The explants incubated for 30 minutes with BrdU at E11.5 were analyzed after increasing time intervals in culture. The BrdU-labeled cells progress from their origin in the rhombic lip towards the floor plate (arrowheads) as schematized in (E). (D) The neurons of the superficial migration express TAG-1 in vitro. (F-G) E12.5 explants incubated for 30 minutes with BrdU maintained in culture for 3 days. TAG-1 transcripts are detected in blue and BrdU immunoreactivity is shown in brown. (F) The TAG-1-labeled cells form thin rows dorsally and arrange in a more diffuse pattern around the ventral midline. (G) Higher magnification of the area framed in F. The arrows point to TAG-1-labeled cells, which have incorporated BrdU at the beginning of the culture period. (H-M) Whole mounts (H,K) and transverse vibratome sections (I,J,L,M) of bulbar explants dissected at E11.5 (H-J) or E12.5 (K-M) and cultured for 3 days in vitro. Except in (J,M), where axons are labeled by immunostaining for L1, the brown label marks the olivary neurons detected by in situ hybridization of Brn3.2 transcripts. In E12.5 explants (K,L), as in vivo, the inferior olives on both sides are separated by the floor plate whereas they fuse ventrally in E11.5 explants (H,I). This behavior fits in with the presence of a ventral gap at the pial end of the floor plate in E11.5 (J) but not in E12.5 (M) explants. Arrowheads, floor plate.

View larger version (107K): [in a new window]   Fig. 3. Consequences of unilateral rhombic lip ablations. (A,B) Explant in which the left rhombic lip was extirpated at the time of explantation (indicated by dots on the right side of the panels). (B) Higher magnification of the area outlined in (A). Some TAG-1- and BrdU-labeled cells have crossed the floor plate. (C,D) Ablation of the left rhombic lip (dots on the right side of the panels) in E11.5 bulbar explants prevents the formation of the ipsilateral inferior olive, indicating that these neurons were located in the dorsal neural plate at the onset of the culture. (D) Vibratome section through the explant illustrated in (C). The olivary neurons cross beneath the floor plate but stop at its contralateral limit. ION, inferior olive; arrowheads, floor plate.

View larger version (67K): [in a new window]   Fig. 4. Influence of the floor plate on the olivary and superficial migrations in vitro. All panels except (E) show bulbar explants dissected at E11.5, cultured for 3 or 4 days, treated for the detection of Brn3.2 transcripts, and viewed from their pial surface. (E) Similar explant dissected at E12.5, cultured for 3 days, and treated for the detection of TAG-1 transcripts. The drawings schematize additional manipulations. The caudal part of the endogenous floor plate was ablated in most cases and both sides of the explants have fused together. (A) Ablation of the floor plate marked in red (F-spondin transcripts) did not prevent the migration of olivary neurons (arrow) towards the midline. (B-D) The olivary neurons accumulate along floor plate transplants dissected from the medulla oblongata (B,D) or the spinal cord (C) of E11.5 embryos. The migration of olivary (D) and superficial (E) neurons is deflected towards a floor plate apposed to the sectioned caudal hindbrain. The inset in E illustrates a higher magnification of the ectopic TAG-1 migration. (F,G) Caudal rhombic lip fragments were transplanted posteriorly (F) or anteriorly (G). They produced Brn3.2+ neurons, which migrated in the ectopic environment (F, spinal cord and G, pons) and accumulated ipsilaterally against the endogeous floor plate. (H,I) Grafts containing floor plates (delineated with dashed lines) were placed at different distances from the rhombic lip (RL). The endogenous midline (asterisk) fused with the additional bulbar tissue. The midline was crossed by olivary neurons but they did not migrate beyond an ectopic floor plate placed closer (H) or farther (I) than the endogenous one. Arrowheads point to the grafted floor plates and arrows to the ectopic ION. Bars, 500 µm (A-E); 300 µm (F-I).

View larger version (56K): [in a new window]   Fig. 5. Chemoattractive and repulsive signalling around the olivary and superficial migrations. Coronal (A,C,D,E,G,I,J) or sagittal (H) sections through the E13.5 caudal hindbrain. (B,F) E11.5 (B) and E12.5 (F) hindbrain explants cultured for 3 and 2 days, respectively, in vitro. (A,B) Netrin-1 midline expression is maintained in E11.5 explants (B) and is similar to its in vivo pattern (A). (E,F) The Dcc netrin receptor is expressed both in vivo (E) and in vitro (F) in the medialmost olivary neurons (arrowheads) and in migrating neurons of the superficial migration (arrows). (C,D,G,H) EphA4 is expressed in the olivary neurons at the end of migration (C) whereas several EphA4 ligands are expressed in the floor plate (D, ephrin B3, ephrin B1 not illustrated) or surround the forming olivary nucleus (G,H, ephrin A5). The inhibitory influence of Sema4C expressing cells (I) could also funnel the olivary migration into its proper pathway. (J) The superficial migration expresses high levels of PACAP transcripts. The arrowheads point to the olivary migration, the arrows to the superficial migration. Asterisks in B, lines of higher netrin expression.

View larger version (41K): [in a new window]   Fig. 6. Influence of netrin on the olivary and superficial migrations. (A-D) Influence of 500 ng/ml purified netrin-1 on hindbrain explants. Netrin promoted profuse axonal outgrowth from rhombic lip fragments grown in collagen (B) compared to control explants (A). The shape of the ION was more regular and more neurons reached the ventral neural tube in hindbrain explants treated with purified netrin (D) compared to untreated control explants (C). (E) E11.5 rhombic lip explant cocultured in collagen gel with netrin (net)-transfected COS cells aggregates (COS-netrin) and control (ctrl) aggregates (En-2-transfected COS cell, COS C). Axons extend from the rhombic lip in the direction of Cos-netrin. Cell migration from the rhombic lip explants is negligible and is not observed before 3 days in culture (data not shown). (F-I) Transplantation experiments similar to those illustrated in Fig. 4D,E. Netrin-transfected (arrowheads) and control COS cells aggregates were used instead of floor plates. Netrin attracts a population of Brn3.2+ (G) and TAG-1+ (H) cells from the dorsal neural tube. I is a detail of H, illustrating the TAG-1+ cells that migrate on the surface of the COS-netrin aggregate.