QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 9 February 2005
doi: 10.1242/dev.01683

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rossel, M. Articles by Carroll, P. Search for Related Content PubMed PubMed Citation Articles by Rossel, M. Articles by Carroll, P.

Reelin signaling is necessary for a specific step in the migration of hindbrain efferent neurons

¹ EPHE Quantitative Cell Biology, INSERM EMI 343, IFR 122, University Montpellier 2, 34090 Montpellier, France
² Laboratoire de Neurobiologie Cellulaire et Moléculaire, UPR 9040 CNRS, 1 Avenue de la Terrasse, 91198 Gif sur Yvette, France
³ INSERM UMR 623, IBDM Campus de Luminy, Case 907, 13288 Marseille Cedex 09, France
⁴ INSERM U.583 Institut des Neurosciences de Montpellier (INM), Hôpital St ELOI, 80 rue Augustin Fliche, 34295 Montpellier Cedex 5, France

View larger version (81K): [in a new window]   Fig. 1. Altered olivocochlear efferent and facial visceral motoneuron development in reeler mutant hindbrain. Motoneuron distribution was determined by using an islet 1 riboprobe on whole-mount preparations. Pial side view of flat-mounted control (A,C,E,G) and reeler (B,D,F,H) embryos at E12.5, E13.5, E14.5 and E15.5. (A) In control embryos at E12.5, islet 1 expression is observed in the Vth (trigeminal motor) and VIIth (facial) migrating nuclei, and in a discrete lateral column of cells (arrows) between these two nuclei that includes a sub-population of the trigeminal, the facial visceromotoneurons (FVM) and the olivocochlear neurons (OC). At this stage many facial motoneurons are still in the process of migrating caudally from their birthplace in rhombomere 4 to their settling position in rhombomere 6. (B) In reeler embryos, VII neuron migration is comparable to control, whereas the OC/FVM column is reduced in its rostrocaudal extension (arrows). (C) In control embryos at E13.5, islet 1 expression is clearly observed in the main Vth nucleus, in the OC/FVM motoneurons, and in the VIIth nucleus. (D) In reeler mutants, the OC/FVM group of motoneurons is barely detectable (arrow). (E-H) At E14.5 (E,F) and E15.5 (G,H), FVM (and/or associated OC) are not labeled by islet 1 at the pial side in reeler embryos (F,H; arrows). At this stage most of the migrations have already occurred or are in the final stages. By E15.5, the facial nucleus forms two lobes, medial and lateral, in control embryos (G, arrowheads). In reeler embryos (H, arrowhead), the medial lobe seems reduced when compared with the control.

View larger version (27K): [in a new window]   Fig. 10. Scheme showing migration defects in the reeler hindbrain. Neuronal population migration is indicated at r4, r5 and r6 levels on the left side of transverse sections of E12.5 control hindbrain. The affected populations are colour coded: VEN/OC, red; FVM, pink; facial branchial motoneurons, light purple; abducens, green. On the right side of the section, reelin expression is indicated. High expression is shown in blue, whereas light expression is represented with blue stripes. Migration defects observed in reeler mutants are illustrated on the right part of the figure. Ventral migration of the OC and FVM neurons is arrested in an intermediate position in reeler mice.

View larger version (95K): [in a new window]   Fig. 2. Reduced and scattered expression of Lhx4 and ER81 by facial MN in reeler mice. The pial side of whole-mount E14.5 control (A,C) and reeler mutant (B,D) mice is shown. (A) Lhx4 is expressed in the trigeminal motor nucleus (V), a column of cells at the r4/r5 level, as well as in the medial part of the facial nucleus (circled), in control embryos. (B) In reeler mutants, the Lhx4-positive area in the facial nucleus is reduced in size (circle), and positive cells were observed deeper in the parenchyme (arrowheads). Most of the rostral column is not present at the pial surface. (C) In control embryos, ER81 mRNA is expressed in cells in the most lateral part of the facial nucleus, and in the inferior olive precursors cells. (D) In reeler mutants, ER81 labeling is mainly localized in the lateral part of the facial nucleus (arrowheads), but in a more scattered pattern than in controls. IO, inferior olive.

View larger version (101K): [in a new window]   Fig. 3. Ectopically positioned motoneurons at r5 level in reeler mutants. (A,B) Vibratome sections after wholemount in situ hybridization with an islet 1 riboprobe at E13.5. Level of the vibratome section is indicated on the whole-mount-stained hindbrain to the right. (A) At r5 level, a compact group of cells is clearly detectable at the pial surface in control embryos (arrow, FVM). (B) In reeler mutants, a strong islet 1 labeling is observed at the ventricular side of the hindbrain (arrow) and a more diffuse group of islet 1-positive cells is localized in a more medial position (arrowhead). Abducens nucleus (ab) is labeled near the ventricular zone. Note the absence of islet 1-positive cells at the pial surface in reeler mutants (circle). (C-E) Vibratome sections after whole-mount in situ hybridization with a Ret riboprobe on control and reeler embryos at E12.5 indicates that ectopic r5 MNs retained FVM markers. (C) Ret is expressed at the end of FVM migration in controls (arrowhead) and in the abducens nuclei near the floor plate (arrow). (D,E) In reeler hindbrains, ectopically positioned Ret-positive cells are observed at levels r4/r5 near the ventricular zone and at intermediate levels (dashed circles), but are absent at the pial surface (circle). v, ventricular surface; p, pial surface. (F,G) Cryostat sections at r5 level of E13.5 embryos labeled with an islet 1 riboprobe. (G) Islet 1 labeling is comparable to results obtained from vibratome sections in reeler mutants: a lack of pial surface labeling (stipple circle), and ectopic groups of cells within the neural tube and close to the ventricular side.

View larger version (103K): [in a new window]   Fig. 4. Aberrant migration of facial visceral motoneurons in reeler mutants. Facial branchial (VII) and visceral motoneurons (FVM) were retrogradely labeled with DiI by injection from the VIIth nerve root at E11.5 and E12.5. Embryos were flat mounted at E11.5 (A,B) and E12.5 (C,D). (A,B) At E11.5, characteristic axon fasciculation and caudal migration of facial motoneurons were observed at the r4 level. White arrow indicates caudally migrating facial motoneurons. In r5, FVMs were migrating from the ventricular zone to a lateral position both in control embryos and in reeler mutants. (C,D) At E12.5, FVMs are organized in a compact group caudal to the axon exit point in control and reeler embryos. (E,F) Transverse vibratome sections of embryos in C and D indicated that FVMs have migrated ventrally toward the pial surface in controls (dashed circle in E). In reeler mutants, FVM neurons remained in a more mediolateral position (circled in F). C-F are confocal images. fp, floor plate.

View larger version (64K): [in a new window]   Fig. 5. Positioning of olivocochlear (OC) precursors is affected in reeler mice. (A) In control embryos at E12.5, Gata3 is expressed in the reticular formation at levels r4-6 and in a dense column of cells positioned rostrally to the facial nucleus and close to the pial surface (circled). (B) In reeler mutants at E12.5, the Gata3 expression pattern was similar to controls except for the dense column of cells (black arrowhead) which was localized on the ventricular side, as visualized in vibratome sections of the same embryos (C-F). Results corresponding to two different levels (r4, C,D; and r5, E,F) are shown. (C,E) A compact group of cells is observed in control embryos at the edge of the reticular formation (arrow). (D,F) In reeler embryos this compact group was absent (circles), but cells were observed in a dorsal position (arrowheads). At the r5 level, Gata3 labeling is observed in a small group of cells at the pial surface (unfilled arrowhead, F; compare with B). (G-L) Retrograde labeling from the VIIIth ganglion (right side) and photoconversion of the DiI signal (brown precipitate) were performed and subsequently followed by Tbx20 ISH (blue precipitate). Photoconversion provided a strong labeling at the VII/VIIIth nerve bundle on two consecutive sections from control embryos (G,H) and reeler mutants (I,K). Using this landmark, we were able to superimpose the ISH (I,K) and fluorescent pictures (J,L). (G,H) At E12.5, Tbx20 is expressed by facial branchial motoneurons (VII), vestibular (VEN) and OC efferents. OC neurons are localized in a ventral position as a compact group (circled). (I,K) In reeler mutants, Tbx20 is strongly expressed in facial motoneurons (red circle) and in a compact group of cells in a dorsomedial position (black circle). No labeling was observed at the ventral side, except for few scattered cells (I, unfilled arrowhead). Comparison with DiI labeling (compare I with J and K with L) indicated that ectopic Tbx20-positive cells accumulate at the position where they should migrate ventrally (dashed circles).

View larger version (70K): [in a new window]   Fig. 6. Normal vestibular efferent location but abnormal medial and lateral olivocochlear efferent neuron localization in the reeler mutant. (A,B) Retrograde labeling of the OC and VEN from the VIIIth ganglion (bilateral injections). Drawings of labeled cells with clear labeled projections and which are not VII precursor cells, were done to clarify injection interpretation. (A) The OCs were identified in a ventral position in control mice. (B) These cells were located ectopically, in close association with VEN, in reeler mutants (circled). (C,D) Retrograde labeling of the OC and VEN from the otocyst (left side) in reeler mutants, to label LOC and MOC cells. Dorsally located cells were labeled on contralateral and ipsilateral sides, which indicated that MOC are affected in reeler mutants (C). A few ipsilateral cells close to the pial surface were labeled on the ipsilateral side only (arrow, D), which should reflect LOC cells migrating to the pial surface. LOC is the major component of the OC precursors, this observation might reflect subpopulation migratory properties, independent of reelin. fp, floor plate; LOC, lateral olivocochlear efferents; MOC, medial olivocochlear efferents; OC, olivocochlear efferent; VEN, vestibular efferent nucleus.

View larger version (79K): [in a new window]   Fig. 7. Relationship between reelin pathway elements and hindbrain neuron ventral migration. Whole-mount ISH for reelin expression revealed an increase in reelin expression between E11.5 and E12.5 (compare A with B). (C-K) Combined reelin ISH followed by islet 1 immunohistochemistry (C,F,I) were performed concomitantly with reelin (D,G,J) and Dab1 (E,F,K) ISH in adjacent vibratome sections at E12.5. (C,D) At r4 level, islet 1-positive OC cells are observed at the pial surface, in close association with a highly reelin-positive area. The circle represents an equivalent position on adjacent sections. (F) At r5 level, islet 1-positive VII motoneurons undergoing caudal migration can be seen near the midline. (G) Reelin-expressing cells are found in several areas with a strong expression near the pial surface. Islet 1-positive FVM cells are observed in a position close to this reelin-positive region (F). (I) At r6 level, VII motoneurons are in the final migratory step toward the pial surface. This shows that the final position of the neurons is a reelin-positive area (J), whereas longitudinal migrating VII motoneurons are reelin negative (F,G). Dab1 is strongly expressed in a region that includes the OC cells in r4 (E), the FVM in r5 (H), and the migrating branchial motoneurons in r6 (K).

View larger version (87K): [in a new window]   Fig. 8. Normal radial glia development in reeler mutant. RC2 (red) and islet 1 (green) labeling in r4 vibratome sections at E12.5. RC2 staining was comparable between control (A) and reeler (B) embryos. Islet 1 labeling indicated the OC ventral position in controls (arrowhead, C) and the ectopic location in reeler mutants (arrowhead, D), together with the facial ventricular location (dashed cicles, C,D). Note the distinct trajectories of the radial glia fibers and the lateral position of the ectopic OC nucleus (arrows, E,F). Autofluorescence of blood vessels was represented in blue (C,D) or pink (E,F).

View larger version (57K): [in a new window]   Fig. 9. Hindbrain reeler-like phenotype for scrambler mutant but no hindbrain phenotype for Reln receptors mutants. (A) At E15.5, islet 1 labels trigeminal motor, OC/FVM and facial motoneurons in control mice. (B,C) Islet 1-labeled OC/FVM cells are absent at the pial surface in reeler (B) and scrambler (C) mice. Abnormal organization of the VII nucleus is seen in reeler and scrambler mutants (lower circle). In double mutants for Reln receptors, ApoER2 and VLDLR, the Isl1 expression pattern is similar to that in control embryos, in particular, the OC/FVM group of cells can be identified at the pial surface (upper circle, D). (E-G) Expression patterns of Dab1, ApoER2 and VLDLR were examined at E12.5 in consecutive vibratome sections through r5 using ISH. Dab1 expression is observed in facial precursors close to the ventricular region (E). ApoER2 was strongly expressed within the neural tube (F); however, VLDLR was only faintly expressed (G). No specific localization in relation to the OC/FVM and facial precursor positions was observed.