1 CNRS UMR C8542, Régionalisation Nerveuse, niveau 8, Ecole Normale Supérieure 46, rue dUlm 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
Author for correspondence (e-mail: wassef{at}wotan.ens.fr)
Accepted 9 October 2001
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SUMMARY |
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Key words: Precerebellar neuron, Tangential migration, Inferior olive, Floor plate, Netrin, Mouse
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INTRODUCTION |
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The floor plate is a complex ventral midline structure involved in neural tube patterning and in the guidance of both growth cone and cell body migration. The floor plate is the source of several short- and long-range chemoattractants and chemorepellents that are likely to govern both the dorsoventral orientation of the olivary and superficial migrations and their different behaviors at the ventral midline. In addition, it could provide olivary neurons with a stop signal preventing them from crossing the midline. Several diffusible and extracellular matrix molecules have been implicated as mediators of the different activities of the floor plate (Kennedy et al., 1994; Serafini et al., 1994; Klar et al., 1992; Echelard et al., 1993; Brose et al., 1999). Netrin-1, which is expressed in the floor plate at all axial levels (Kennedy et al., 1994) and contributes to the attractive action of the floor plate on growing axons (Tamada et al., 1995; Shirasaki et al., 1995; Shirasaki et al., 1998), has been implicated as a major chemoattractant for ION (Bloch-Gallego et al., 1999) and LRN neurons (Alcantara et al., 2000). In netrin-1 mutant mice the ION neurons are misplaced, their number is reduced and their cerebellar projection is abnormal (Bloch-Gallego et al., 1999). Retrograde tracing experiments suggest that the LRN neurons are less affected in netrin1 mutants (Bloch-Gallego et al., 1999). The developing ION and LRN neurons express several netrin receptors of the Dcc and Unc-5 families (Ackerman et al., 1997; Bloch-Gallego et al., 1999), which is consistent with a direct role of netrin-1 on their dorsoventral migration.
In the present study we examined directly the influence of the local environment on the behavior of neurons of the olivary and superficial migrations, identified by Brn3.2 and TAG-1 expression, respectively. We compared their responses in bulbar explants when grafted in an ectopic location, to those ectopic fragments of midline structures or to sources of diffusible netrin-1 protein.
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Materials and Methods |
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Lightly ether-anaesthetized pregnant mice were injected in the tail vein with a solution of BrdU (Sigma; 2 mg/ml; 20 µg/g) in 0.9% NaCl on gestational days 10.5, 11.5 and 12.5 to identify neuronal birthdates. Embryos were fixed at E13.5, E14.5 or E18.5. DNA-incorporated BrdU was detected by immunocytochemistry.
Immunocytochemistry
Dissected hindbrains or explants were fixed overnight at 4°C in F4% for the detection of e-NCAM, L1 (gifts from Dr Schachner), vimentin (Amersham) and BrdU (Becton Dickinson). Immunocytochemistry was performed on vibratome (100 µm thick) or on cryostat (14 µm thick) sections of hindbrain or on whole explants. Sections or explants were incubated with the first antibody for 48 hours at 4°C, revealed with a biotinylated secondary antibody (1/300, Jackson) for 1 hour at room temperature followed by streptavidin-biotin peroxidase (1/400, Amersham) and visualized with diaminobenzidine and H2O2. For the detection of incorporated BrdU, vibratome sections or explants were treated with 2 N HCl for 45 minutes, and digested for 10 minutes with 20 µg/ml proteinase K. The sections were postfixed in PFA4% containing 0.2% glutaraldehyde and treated as above.
In situ hybridization
E11.5 to E15.5 embryos or explants at the end of the culture period were fixed overnight at 4°C in F4%. Transverse vibratome sections (200 µm thick) of albumin-gelatin embedded dissected hindbrains or fixed explants were dehydrated in methanol and stored at 20°C. In situ hybridization and double-colour in situ hybridization were performed on rehydrated tissue as described (Bally-Cuif and Wassef, 1994). The diluted anti-digoxigenin antibody was adsorbed overnight on a confluent layer of EBNA-293 cells before being used in experiments involving COS or EBNA cells, as it was found to crossreact with an epitope present in variable amounts on these cells.
Probes
The mouse Brn3.2 template was generated by PCR from mouse genomic DNA based on the published sequence (Turner et al., 1994). A 524 bp fragment between nucleotides 548 and 1071 was amplified and subcloned in pBS. The entire coding region of rat TAG-1 cDNA (Furley et al., 1990) was provided by Dr T. Jessell. pBS plasmid containing sequences from the 3' UTR region of mouse netrin was provided by Dr T. Serafini. Plasmids containing cDNA fragments of rat F-spondin (gift from Dr Klar), mouse PACAP (gift from Dr Waschek) EphA4, ephrin A5, ephrin B1 and B3 (gift from Dr Gilardi) and SemaF (gift from Dr Chedotal) were also used.
Culture procedures
Preparation of the explants
Explants containing the whole rhombencephalon including the cerebellum were dissected from E11.5 and E12.5 brains in PBS-0.6% glucose, and opened on the dorsal midline. They were cultured for 1-4 days ventricular side down on Biopore membranes (Millipore) floating on DMEM/F12 medium (Gibco) supplemented with antibiotics and with 10% fetal bovine serum and 5-10% horse serum. In some experiments the medium was supplemented with 500 ng/ml of purified chick netrin-1 (gift from Dr Tessier Lavigne) or with BrdU (10 mM, for 30 minutes).
Ablations and transplantations
Fragments of floor plate, rhombic lip or ganglionic eminence were ablated or dissected using tungsten needles and scalpel blades. Transplants were grafted at different levels onto hindbrain explants. Alternatively, hindbrain explants were cut transversally in the region of migration and a floor plate explant or a COS or EBNA-293 cell aggregate was placed against the cut edge. Fragments of E11.5 cerebral cortex or ganglionic eminence were inserted into the region of migration. The transplantation procedure is outlined on the side of each panel.
Unless otherwise specified, the explants were cultured for 3-4 days in vitro, fixed overnight in PFA4%, and processed for whole-mount in situ hybridization or immunocytochemistry.
Sources of netrin protein
COS7 cells were transfected with pGNET1myc or pGNET2myc (using Lipofectamine, Gibco BRL) as described by Serafini et al. (Serafini et al., 1994), or using Fugene 6 (Boehringer), according to the manufacturers instructions. pTLmEn2m (Joliot et al., 1998) was used as an unrelated control plasmid. An EBNA-293 cell line stably transfected with a netrin-1 expression plasmid and the control line transfected with the empty vector were also used. Aggregates of EBNA or transfected COS cells were prepared by the hanging drop method (Kennedy et al., 1994) and used as transplants in the same way as tissue fragments. Restricted sources of purified netrin were established in different ways. Affigel beads were rinsed in PBS and incubated overnight with a solution of netrin-1 (5 µg/ml in DMEM/F12) and used as transplants. Low-melting-point agarose in DMEM/F12 was mixed with a solution of purified chick netrin-1 (10 µg/ml final) and some was aspirated into gel-saver tips. After cooling, the blocks or tips were cut into smaller pieces, which were used as sources of netrin-1 protein. BSA was used instead of netrin in control experiments. As a biological test of netrin-like activity, the putative sources of netrin-1 were cocultured in collagen gels at a small distance from rhombic lip fragments and axonal outgrowth was examined at 24 and 48 hours.
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RESULTS |
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At E12.5, Brn3.2 transcripts were expressed in a circumferential band of cells abutting the rhombic lip and located deeper in the parenchyma than the superficial migration (Fig. 1H). The Brn3.3 and TAG-1 positive cells occupied complementary domains in the subventricular layer. At E13.5, the Brn3.2 positive cells gathered in the ventral half of the neural tube (Fig. 1I), in a domain corresponding to the olivary migration (Altman and Bayer, 1987a; Turner et al., 1994), which avoided the floor plate. At E14.5 the Brn3.2 signal was concentrated in the ION (Fig. 1J). Some dorsally located neurons also expressed Brn3.2 at this stage. Later on, Brn 3.2 expression persisted only in the ION. TAG-1 and Brn3.2 were used as markers of the two migrations in subsequent experiments.
BrdU injections were used to determine the birthdates of the neurons of the superficial and olivary migrations more precisely than was possible in earlier experiments (Taber Pierce, 1973) (data not shown). The bulk of olivary cells was generated at E10.5, with a smaller population labeled by BrdU injections at E11.5. The BrdU-labeled olivary neurons reached the ventral midline at E13.5. The peak of LRN and ECN neurons production occured 1 day later, at E11.5. BrdU injections at E12.5 also resulted in the labeling of some neurons in these nuclei. The BrdU-labeled neurons of the superficial migration reached the ventral hindbrain by E13.5.
The superficial and olivary migrations in bulbar explants
To examine whether the bulbar migrations could resume in vitro, explants containing the hindbrain including the cerebellum were dissected from E11.5 or E12.5 mouse embryos and cultured for 1-8 days, ventricular side down, in an open book configuration.
A 30-minute pulse of BrdU at the beginning of the culture period was used to label the superficial migration. The explants were fixed after 1-3 days in vitro and treated in toto for BrdU immunocytochemistry. In this way, only the most superficial cells of the ventricular and pial surfaces were accessible to the antibodies. The BrdU-labeled cells, which were confined to the vicinity of the rhombic lip after 1 day in vitro (Fig. 2A), progressed towards the midline during the two following days (Fig. 2B-E). 3 days after explantation they formed a sharply delimited band continuous across the midline (Fig. 2C,E). Detection of BrdU or TAG-1 (Fig. 2D) transcripts revealed similar migration patterns on explants dissected at E11.5 (Fig. 2C,D) or E12.5 (Fig. 2F,G). Thus, the migration of cells of the superficial stream proceeded normally in vitro even if slightly slower than in vivo. Further maturation did not proceed in vitro: the band of TAG-1+ or BrdU+ cells did not disappear even after 8 days in culture (data not shown) and the explants maintained the same appearance as at 3 days in culture.
In E11.5 explants, Brn3.2 was detected in two longitudinal dorsal stripes, unrelated to the ION, and extending along the whole hindbrain. After 2 days in vitro, the ION was detected as an isolated medial patch of Brn3.2 expressing (Brn3.2+) cells in the caudal hindbrain. 1 day later, these cells reached the ventral midline, where they accumulated (Fig. 2H). In most explants, a faint labeling for Brn3.2 was also detected in the floor plate region (Fig. 2H). In transverse sections of the explants, the Brn 3.2+ ION was located more ventrally than in vivo and was fused on the midline under the floor plate (Fig. 2H,I). In explants dissected at E12.5 instead of E11.5 and maintained for 3 days in culture, Brn3.2 transcripts were detected in the two ION on both sides of the floor plate but never across (Fig. 2K,L). More commissural axons were detected on transverse sections of E12.5 explants immunostained for L1/Ng-CAM (Fig. 2M) compared to similar explants dissected at E11.5 (Fig. 2J). In addition, no gap was detected between the floor plate and the pial surface of the explant (compare Fig. 2J,M).
Origin of the TAG-1 and Brn3.2 neurons in the rhombic lip and their behavior at the floor plate
Unilateral ablation of the rhombic lip at the beginning of the culture, at E11.5 (n=8) or E12.5 (n=12), resulted in a sharp decrease in the number of dorsal TAG-1+ or BrdU neurons on the operated side (Fig. 3A,B). After 4 days in culture, TAG-1+ neurons that have crossed the midline were detected ventrally on the operated side (Fig. 3A,B).
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Taken together, these observations indicated that the superficial and olivary neurons migrated in vitro, although some alterations could be detected after 3 days in culture. Unless otherwise specified, in the experiments described below, ablations and transplantations were performed at E11.5 and the bulbar explants were cultured for 3 or 4 days before being fixed.
The total number of transplanted or ablated explants was over 950. Slightly more than one third were discarded because of contamination, inefficient transfection, high background staining or poor histochemical reaction, which were the main causes of variability in these experiments. In two thirds of the remaining explants, the experimental design was a variant of that described here, i.e. comparison of the effects of COS-netrin and floor plate on the same explant instead of COS-netrin and COS-control, graft of rosa 26 rhombic lip instead of wild type, etc. The numbers of explants that were treated in the experiments (n) are indicated.
Influence of the floor plate on the olivary and superficial migration
The floor plate was ablated in the region of migration at the beginning of the culture period (Fig. 4A-E). In some cases both sides of the explants fused together on the midline during the culture period. The completion of the ablation was checked using a rat F-spondin probe as a floor plate marker (n=9, Fig. 4A). In most cases (8/9), the Brn3.2+ cells reached the ventral midline but failed to form a central compact structure (Fig. 4A; compare with Fig. 2H). The TAG-1+ neurons also migrated to the ventral edge of the explant in the absence of a floor plate structure (3/3, arrow in Fig. 4E). These observations indicated that, at the onset of their migration, the superficial or olivary neurons do not any longer require the presence of a floor plate to reach the ventral neural tube.
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The signals necessary for olivary migration are widely distributed in the caudal brain
To examine the role of intervening structures in the migration of olivary neurons, rhombic lip fragments were dissected from the caudal hindbrain and transplanted in various ectopic sites: in the most dorsal part of the spinal cord (n=4, 4/4, Fig. 4F) or in the pontine rhombic lip (n=4, 3/4, Fig. 4G). The endogenous floor plate was left in place. After 3 days in culture, a group of Brn3.2+ cells was detected close to the endogenous floor plate ipsilaterally to the grafted rhombic lip. No Brn3.2 staining was detected near the floor plate on the contralateral side. Thus, although the migration domain of olivary cells is delimited in vivo as well as in control explants, these cells were capable of migrating in ectopic environments either in the rhombencephalon or in the spinal cord. In addition, the Brn3.2+ cells always remained ipsilateral to the rhombic lip grafts (Fig. 5C). In contrast, no ectopic TAG-1 positive cells were produced by similar grafts.
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Together with the results described above, these observations indicate that the floor plate not only attracts olivary cells during migration but also provides them with a short-range stop signal. Signals from the ventral neural tube could also contribute to slowing down migration as the olivary neurons reach their final destination.
Chemoattractive and repulsive candidates that could mediate the ventral influences on the migrations
Netrin-1 and its Dcc and Unc-5 family receptors are expressed in the caudal hindbrain during the period of migration (Bloch Gallego et al., 1999; Alcantara et al., 2000). We examined whether the pattern of expression of netrin-1 and Dcc was maintained in hindbrain explants. In E11.5 hindbrain explants maintained for 3 days in vitro, netrin-1 was expressed in the medial region of the explants with a peak of expression at the floor plate, and another more laterally (Fig. 5B), which was consistent with their in vivo pattern (Fig. 5A). In E12.5 explants maintained for 2 days in vitro, Dcc was expressed in a thin medial stripe at the level of the ION and, more laterally, in neurons of the superficial migration (Fig. 5F). This pattern is consistent with the in vivo expression of Dcc in the medial olive and the superficial migration at E13.5 (Fig. 5E).
EphA4 was expressed in the olivary migration at E13.5 (Fig. 5C). At the same stage, EphA4 ligands were expressed both in the floor plate (ephrin B3, Fig. 5D; ephrin B1, similar pattern, not shown) and surrounding the olivary migration (ephrin A5, Fig. 5G,H). An inhibitory ensheathing could also be provided by cells expressing Sema4C (Fig. 5I). The superficial migration neurons expressed high levels of the pituitary adenylyl cyclase activating peptide (PACAP), which could modulate various aspects of their intracellular signalling.
Influence of netrin on the olivary and superficial migrations
The influence of ubiquitous or local overexpression of netrin-1 on the migration of olivary and superficial neurons was examined. E11.5 hindbrain explants were incubated for 30 minutes in culture medium containing (or not) purified chick netrin-1 protein (500 ng/ml), a concentration that elicited a robust axonal outgrowth from rhombic lip explants (compare Fig. 6A and B). After 3 days in culture, the shape of the ION was more regular and compact near the floor plate in netrin-treated explants (n=7,5/7, Fig. 6D) compared to control explants (n=6, Fig. 6C).
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DISCUSSION |
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Influence of the local environment on the olivary migration
Floor-plate ablations did not prevent the migration of olivary neurons towards the midline, showing that local cues that could guide dorsoventral migration were already distributed in the neural tube at E11.5. The presence of a repulsive activity in the rhombic lip was unlikely because many olivary neurons succeeded in reaching the ventral midline in experiments where dorsal neural tube ablations were too narrow. Olivary neurons migrated out of rhombic lip fragments transplanted at more anterior levels of the hindbrain or more posteriorly in the spinal cord, indicating that their migration is not dependent upon caudal hindbrain specific cues. At this stage of development, signals capable of maintaining the motility of olivary neurons or providing them with a permissive substrate are widely distributed along the caudal neural tube. However, these cues are not ubiquitous, as Brn3.2 neurons failed to migrate from rhombic lip explants cocultured with cerebral cortex tissue or in collagen gel or matrigel (I. dD. and M. W., unpublished observation).
Inhibitory signals to olivary migration in the ventral neural tube
Olivary neurons migrating in the spinal cord or the pons always ended as a tight cluster against the floor plate. This contrasts with the diffuse and wider distribution of olivary neurons in the caudal hindbrain. This behavior is even enhanced when supernumerary ventral tissue is inserted in the olivary migration pathway, as illustrated in Fig. 4I. This suggests that cues present in the ventral domain either inhibit olivary migration or interfere with cell-cell adhesion modifications that are necessary for their condensation into distinct nuclei. Several candidate molecules that could mediate the inhibitory behavior of the ventral neural tube on olivary migration have been described. We have observed that ephrin A4 is expressed in the inferior olive at E13.5, whereas several putative ligands are expressed around the olivary migration (ephrin A5), in the floor plate (ephrin B1 and B3) or slightly later between the olivary lamellae (ephrin A5 at E15). The ventral part of mouse rh7/8 contains a large patch of Sema3A expressing cells (Sem D) (Varela-Echavarria et al., 1997) and several small patches of neurons expressing Sema4C line the olivary migration. The olivary neurons, on the other hand, have been reported to express low levels of the neuropilin 2 semaphorin receptor (Chen et al., 1997) but not neuropilin 1, a selective receptor of Sema3 family members (Kawakami et al., 1996). This complex, though still lacunary, network of inhibitory influences is probably deployed in order to delimit a wide mediolateral outline of the mature ION where the olivary migration settles and begin the complex rearrangements that accompany the morphogenesis of the characteristic ION subdivisions (Bourrat and Sotelo, 1991; Wassef et al., 1992).
The floor plate provides a stop signal to olivary neurons
Two known properties of the floor plate could account for the behavior of olivary neurons whose cell bodies stop at the floor plate, in contrast to their axons, which cross it. On the one hand, the tightly packed radial glia of the midline could behave more like a mechanical obstacle to cell bodies than to axons. Nevertheless, in E11.5 explants, the olivary cells reach the pathway of the superficial migration and find their way beneath and across the floor plate, possibly as a result of the elimination of longitudinal axon tracts in the explants. These neurons are not capable, however, of pursuing their migration on the other side. The floor plate has been shown to act as a switch that changes the localization of adhesion molecules and guidance receptors from internal pools to the cell surface, and vice versa, as growth cones cross the midline (Dodd et al., 1988; Stein and Tessier-Lavigne, 2001). As the olivary neurons cross beneath the floor plate they could similarly receive a short range signal from the floor plate preventing them from migrating contralaterally.
Influence of netrin on the olivary migration
The influence of netrin-1 on the olivary migration was tested in various ways. Purified netrin was diluted in the culture medium or diffused from focal sources; aggregates of netrin-transfected COS cells or a netrin-secreting EBNA-293 permanent cell line served as netrin sources. Netrin had two clear influences: it attracted a restricted subpopulation of Brn3.2-expressing neurons from the dorsal neural tube and, when provided in the culture medium, facilitated migration, resulting in a smoother and more regular outline of the ION nucleus near the floor plate. On the other hand, netrin sources did not mimick floor plate fragments that attracted and stopped Brn3.2 neurons when inserted into their pathway or placed against the edge of hindbrain explants cut in the region of migration. Compared with the phenotype of netrin mutants, the modest influence of exogenous netrin on the in vitro olivary migration is puzzling. We have observed that dorsoventral migration proceeds normally in vitro when the floor plate is ablated at the onset of migration, suggesting that, in the absence of a high ventral source of netrin, the olivary migration relies on the dorsoventral distribution of hindbrain cues. In netrin mutants, besides the lack of a ventral source of netrin, the dorsoventral organization of the neural tube is probably disturbed before the onset of olivary migration, which could explain the ectopic distribution of clusters of olivary neurons around their normal path. In addition, the direct influence of netrin on migrating olivary neurons could be restricted in time. Dcc transcripts are detected at both ends of the migration but not in migrating olivary neurons. At one end, premigratory olivary neurons in transit in the subventricular zone probably transiently express Dcc transcripts. At the other end, at E13.5, the medial subset of olivary neurons that abuts against the floor plate contains Dcc transcripts. The persistence of the DCC protein in the migrating ION neurons is unknown. COS-netrin cells were only active when placed near the dorsal neural tube, suggesting that the DCC protein is short-lived. On the other hand, we find that addition of soluble netrin to the medium globally improves the migration on ION neurons in vitro, especially in the ventral domain. In these experiments, netrin could influence both the early and late phases of olivary migration, allowing ION neurons to reach the ventral domain before the complete downregulation of DCC protein expression. At subsequent stages of development, Dcc becomes widely expressed in the ION (Bloch-Gallego et al., 1999) and could affect axonal pathfinding.
Influence of the local environment on the superficial migrations
As observed for olivary migration, floor plate ablation did not prevent the superficial migration from reaching the ventral hindbrain. Similarly, rat commissural axons still project ventrally in the absence of floor plate (Placzeck et al., 1990). In contrast to olivary migration, however, establishment of the superficial migration was constrained by the continuity of structural elements, as it did not cross the limit between grafted and host tissue in homo- or heterotopic rhombic lip transplantation experiments. We have recently observed (Kyriakopoulou et al., 2002) that TAG-1 homophilic binding is essential for the migration of superficial neurons in explants. It is likely, however, that other environmental cues are important for the superficial migration. Whereas we find that TAG-1+ cells from the explants are attracted by COS-netrin aggregates and we could confirm that netrin-1 elicited a robust outgrowth of TAG-1+ axons from E11.5 or E12.5 caudal rhombic lip explants cultured in matrigel or collagen gels (Alcantara et al., 2000), very few cells migrated on these axons during the first 3 days in culture. In several instances, glial cells were found to provide a blueprint prefiguring neuron migration pathways: astrocytic sheathing wraps the migrating olfatory neuron precursors (Lois et al., 1996) and loose glial processes prefigure the pontine migration pathway (Ono and Kawamura, 1990). Glial end-feet tunnels, similar to those observed beneath the floor plate, could be a prerequisite for the superficial migration. Meninges, which are removed at the onset of migration, could also be a source of signals, as they are for external granule cells (Ma et al., 1998). We find that TAG-1+ cells prematurely stop migration after 2 days in vitro in our explant system.
Influence of netrin on the superficial migration
We observed that netrin attracts the TAG-1 expressing cells of the superficial migration and that these neurons express Dcc. It was therefore unexpected that the LRN apparently forms and projects normally to the cerebellum in netrin-1 mutants (Bloch-Gallego et al., 1999). We observed that PACAP is expressed at high levels in the superficial migration, whereas the PAC1 receptor has been reported to be expressed ubiquitously in the hindbrain (Washek et al., 1998). A high cytoplasmic concentration of cAMP could increase the sensitivity of the neurons of the superficial migration to a residual netrin signal present in the mutants, or to signalling by another Dcc ligand or cofactor (Ming et al., 1997). Similar to the neurons of the superficial migration, the pontine neurons respond to netrin signals from the onset of their migration but are nevertheless able to reach the ventral pons in netrin mutants (Yee et al., 1999).
The present study, through the use of an organotypic system that enabled direct manipulation of the olivary and superficial migrations of the caudal hinbrain, has assessed more precisely the relative importance of structures and molecular signals that influence the migration of these population of precerebellar neurons.
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ACKNOWLEDGMENTS |
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