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First published online February 24, 2006
doi: 10.1242/10.1242/dev.02283
1 Laboratory of Neuroscience, Graduate School of Frontier Biosciences, Osaka
University, Suita, Osaka 565-0871, Japan.
2 SORST, Japan Science and Technology, Kawaguchi, Saitama 332-0012, Japan.
3 Division of Behavior and Neurobiology, National Institute for Basic Biology,
Myodaiji-cho, Okazaki 444-8585, Japan.
4 Department of Development and Differentiation, Institute for Frontier Medical
Sciences, Kyoto University, Kyoto 606-8507, Japan.
Author for correspondence (e-mail:
murakami{at}fbs.osaka-u.ac.jp)
Accepted 13 January 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Neuronal migration, Precerebellar neurons, Nucleogenesis, In vivo electroporation, Midline crossing, Ventricle-directed migration, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mechanisms in the development of stratified structures have been well
documented (reviewed by Hatten,
1999
; Marin and Rubenstein,
2003; Komuro and Yacubova,
2003; Bielas et al.,
2004). However, much less is understood about nucleogenesis.
Recently, it has been proposed, in the midbrain, that periodic patterns of
molecularly distinct stripes that appear to be induced by sonic hedgehog and
FGF8 play an important role in the formation of the red nucleus and oculomotor
nucleus (Agarwala and Ragsdale,
2002). Concentration-dependent induction by morphogens such as
sonic hedgehog and FGF8 may explain the formation of laminated or striped
structures (reviewed by Goulding and
Lamar, 2000), but cannot fully explain the emergence of discretely
distributed nuclei.
Neuronal migration is another potential factor in nucleogenesis and its
role is well established in the formation of stratified structures. In the
cerebral cortex, for example, excitatory neurons that are born in the
ventricular zone migrate along the radial glial cells that span the cerebral
wall (Rakic, 1972;
Hatanaka and Murakami, 2002)
(reviewed by Kriegstein and Noctor,
2004) and settle in specific lamina in a birthdate-dependent
manner (Rakic, 1974).
Later-born cells migrate past earlier-born cells, forming inside-out laminated
structures that lie parallel to the pial surface
(Angevine and Sidman, 1961;
Berry and Rogers, 1965).
Here, we examined how neuronal migration leads to establishment of nuclei,
focusing on the hindbrain where clear nuclear organization exists. A number of
studies have addressed the migration of hindbrain neurons and several genes
affecting their migration have been identified (reviewed by
Hatten, 1999;
Wingate 2001;
Chandrasekhar, 2004;
Sotelo, 2004). Yet, cellular
basis for nucleogenesis is still poorly understood. Specifically, how neurons
with common properties accumulate in a defined position remains unknown. We
have chosen mossy fibre-projecting precerebellar (PC) nuclei as a model,
because their origin and development is well documented
(Altman and Bayer, 1997;
Rodriguez and Dymecki, 2000;
Wang et al., 2005). Previous
studies have suggested that neurons of the PC nuclei are derived from the
lower rhombic lip (lRL), the germinal neuroepithelium surrounding the alar
recess of the fourth ventricle (Harkmark,
1954; Taber-Pierce,
1966; Tan and Le Douarin,
1991; Altman and Bayer,
1997; Rodriguez and Dymecki,
2000; Wang et al.,
2005), and migrate circumferentially. Although these studies
examined the origin, migratory route and the fate of alar plate-derived
neurons, the cellular processes leading to nucleogenesis were not established,
owing to the lack of techniques that allow in vivo visualization of individual
neurons migrating from a specific site of the neural tube. To clarify these
issues, we introduced the enhanced yellow fluorescent protein (EYFP) gene into
the lRL of mouse embryos and visualized migration and nucleogenesis of mossy
fibre-projecting PC neurons.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression vectors
The whole coding regions for EYFP (Clontech) and Venus
(Nagai et al., 2002) were
cloned into the pCAGGS vector (Niwa et
al., 1991), yielding pCAG-EYFP
(Saba et al., 2003) and
pCAG-Venus, respectively. For inhibition of cadherin function, pCAG-Ncad(t)
that carried a gene encoding an intracellular domain of N-cadherin
fused to enhanced green fluorescent protein (EGFP) was
prepared (H.T, D.K., K. Nishida and F.M., unpublished). A vector carrying
Venus was kindly provided by Dr A. Miyawaki (BSI, Riken).
Labelling of PC neurons by exo utero electroporation
Exo utero electroporation was performed as previously described
(Saba et al., 2003)
(Fig. 1A) with some
modifications. In brief, plasmids were diluted in 0.01% Fast Green or Indigo
carmine (final concentration, 
2 µg/µl) to monitor the injection and
was injected into the fourth ventricle of an embryo exposed by cutting the
uterine wall.
Three electric pulses (20 V, 50 ms) were applied with an electroporator (ECM830, BTX). Plasmids were unilaterally introduced into the lRL of E12.5 mouse embryos. The vector, once incorporated into the ventricular neuroepithelial cells, should be present in their descendents. Embryos with unilaterally labelled lRL were selected by epifluorescence microscopy (SZX12, Olympus). Hindbrains were removed from these embryos and immersed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer at pH 7.4 (4% paraformaldehyde) overnight at 4°C. The fixed hindbrains were imaged using a cooled CCD camera equipped with epifluorescence illumination (VB-G25, Keyence). Alternatively, the hindbrains were embedded in OCT compound (Sakura Finetechnical) after immersion in 30% sucrose in PBS. Sections (18-20 µm) were cut on a cryostat (HM-500, Zeiss) and mounted on slides (12-550-15, Fisher Scientific). The distribution of EYFP- or Venus-labelled cells was analysed using a confocal microscope (TCS SP2 AOBS, Leica).
In situ hybridization
The vector carrying the Mbh2 gene (AB043980; GenBank) was
linearized and used as a template for RNA synthesis. An RNA probe for
Mbh2 was subsequently synthesized using a digoxigenin (DIG)-11-UTP
mixture (Roche) and T3 or T7 RNA polymerase (Roche), and purified with Quick
spin columns (Roche).
Cryosections from the hindbrain of E14.5-18.5 mouse embryos prepared as described above were dried at 50°C for 3-5 hours. The dried sections were refixed in 4% PFA for 5 minutes, and then treated with 0.3% hydrogen peroxide in PBS for 30 minutes. After acetylation with 0.25% acetic anhydride in 0.1 M triethanolamine, the sections were probed with DIG-labelled Mbh2 cRNA probes (2 ng/µl) in hybridization solution (50% formamide, 5x SSPE, 5% SDS, 200 µg/ml yeast tRNA) for 16 hours at 70°C. After washing, the sections were treated with RNase A (20 µg/ml; QIAGEN) for 1 hour at 37°C. Samples were reacted with a peroxidase-conjugated anti-DIG antibody (1:200, Roche) in Tris-buffered saline containing 1% blocking reagent (Roche). The tyramide signal amplification system (PerkinElmer Life Sciences) was then applied following the manufacturer's instructions.
Immunostaining
A rabbit polyclonal anti-green fluorescent protein (GFP) antibody (1:1000,
Molecular Probes) was used on sections of EYFP- and Venus-labelled hindbrain
tissues. To detect EYFP/Venus denatured by in situ hybridization procedures,
the sections were reacted with the anti-GFP antibody for 2 hours at room
temperature. After a washing with 0.1% Tween-20 in PBS, the signal was
visualized with an Alexa488-conjugated anti-rabbit IgG antibody (Molecular
Probes).
Radial glial fibres were identified using an anti-nestin antibody (rat401)
(Hockfield and McKay, 1985).
The antibody was diluted 1:5 in 0.3% Triton-X100 in PBS (PBST) and applied to
30-40 µm sections that had been pretreated with 0.3% hydrogen peroxide in
PBS for 30 minutes at room temperature. After overnight incubation at room
temperature, sections were rinsed in 0.3% PBST and reacted with biotinylated
anti-rat IgG antibody (1:200, Jackson ImmunoRes). Sections were then treated
with Vectastain Elite ABC kit (PK-6100, Vector) followed by the tyramide
signal amplification method as described above.
BrdU labelling study
For birthdating studies, intraperitoneal injections with
5'-bromo-2'-deoxyuridine (BrdU) (Sigma; 5 mg/ml in sterile saline)
were made in EYFP-electroporated pregnant mice at 50 mg/kg body
weight to label embryos at E12.5, E13.5 and E14.5 (n=5 at each
stage). To detect incorporated BrdU, 25 µm transverse sections of the pons
were prepared. These sections were incubated in 4 N HCl for 45 minutes, rinsed
in 0.2% PBST and reacted with a rat monoclonal anti-BrdU antibody (Cat#
OBT-0030, 1:200, Oxford Biotechnology) for 2 hours at room temperature. After
extensive washing with 0.2% PBST, the sections were incubated with a
biotinylated anti-rat IgG antibody (1:200, Jackson ImmunoRes) for 2 hours at
room temperature followed by visualization with Alexa594-conjugated
streptavidin. Subsequently, the EYFP protein that had been denatured by the
HCl treatment was stained with an anti-GFP antibody.
Captured images were thresholded for quantification using Leica confocal software (Ver 2.61). The total number of EYFP-labelled cells (Ntotal) and the number of EYFP-labelled cells that were also positive for BrdU (NBrdU) were counted in the entire PGN/RTN on the side of the contralateral to the DNA injection. The ratio of BrdU-positive cells to all EYFP-labelled cells (NBrdU/Ntotal) was calculated.
Electron microscopy
The hindbrain of E15.5 mice was removed and immersed in a fixative
consisting of 4% PFA (TAAB) and 0.2% glutaraldehyde (EM Science) in 0.1 M
sodium cacodylate (Nacalai Tesque). The preparation was further fixed in 2%
glutaraldehyde at 4°C overnight followed by postfixation in 2% OsO4 and
then subjected to conventional procedures of electron microscopy. Thin
sections were cut coronally. The plane of section was carefully chosen so that
radially running fibres can be observed in the sections. Electron microscopy
was carried out in Hanaichi Denshi Kenbikyo Laboratory.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PC neurons can be specifically labelled in vivo by exo utero electroporation of EYFP
The neurons of the external cuneate nucleus (ECN) and the lateral reticular
nucleus (LRN) are generated earlier (E10.5-13.5) than those of the pontine
grey nucleus (PGN) and reticulotegmental nucleus (RTN) (E11.5-16.5)
(Taber-Pierce, 1973). The
former migrate circumferentially to give rise to the posterior extramural
migratory stream (PEMS), while the latter take a more anteriorly oriented
route to form the anterior extramural migratory stream (AEMS)
(Altman and Bayer, 1987a;
Altman and Bayer, 1987b).
Direct visualization of lRL-derived cells largely confirmed these previous
findings (Fig. 1B,C;
n=16). Fig. 1B shows
the dorsal view of the E14.5 mouse hindbrain expressing EYFP in the right lRL.
A rostroventrally directed stream of EYFP-labelled cells emerged from the lRL.
Migrating cells that formed the PEMS were barely visible in the dorsal
hindbrain at this stage, perhaps because they had already emigrated (arrowhead
in the inset of Fig. 1B).
Indeed in the ventral view, neurons in the PEMS are clearly visible
(Fig. 1C, arrowhead marked
PEMS). These observations show that cells leaving lRL take two specific
pathways: one towards the metencephalon and the other towards the
myelencephalon.
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EYFP-labelled cells migrated immediately beneath the pial surface
(Fig. 1D,E, left panels). These
observations, together with those from whole-mount preparations, strongly
suggest that the labelled cells are PC neurons. To characterize the
EYFP-labelled cells further, we examined the expression of
Mbh2/Barhl1, a Bar-class homeobox gene expressed by PC neurons that
project mossy fibres, and disruption of Mbh2 causes malformation of
PC nuclei (Li et al., 2004).
We found that Mbh2 was expressed by most of the EYFP-labelled cells
in the AEMS (Fig. 1D) and PEMS
(Fig. 1E).
Migration and subsequent nuclear formation of ECN/LRN neurons
EYFP-labelled cells in the PEMS migrate across the midline
We observed midline crossing of future ECN/LRN neurons, consistent with
previous observations (Bourrat and Sotelo,
1990; Altman and Bayer,
1987a). At E14.5, many EYFP-labelled cells were found in the
ipsilateral myelencephalon, migrating tangentially along the dorsal rim of the
myelencephalon (Fig. 2A;
n=12); they had extended leading processes and were aligned in an
orderly fashion, forming several thin layers
(Fig. 2B). In the ventral
midline region, some EYFP-labelled cells extended their leading processes
towards the contralateral side (Fig.
2C and inset). These morphological features, together with the
absence of labelled cells occupying other regions on the contralateral side,
demonstrate that these cells cross the midline.
At this stage, EYFP-labelled neurons did not form aggregates on their ipsilateral migratory route. However, clustered expression of Mbh2 signal was recognized in the LRN region (Fig. 2D). This is probably due to early-born LRN neurons originating from the contralateral lRL, and suggests that mechanisms for nucleogenesis already operate at this stage. Therefore, the failure of ipsilaterally migrating EYFP-labelled neurons to form aggregates in this location (Fig. 2D) may be due to immaturity of these migrating neurons, rather than to underdeveloped environmental cues required for nucleogenesis.
A change in migratory directions from tangential to radial
Between E15.5 and E16.5, clusters of labelled cells formed in two distinct
regions of the contralateral medulla corresponding to the ECN and LRN
(Fig. 2E,G; n=12)
(Altman and Bayer, 1987a). Some
of the cells in these clusters were found some distance beneath the pial
surface, and had extended radially oriented processes
(Fig. 2F,H). These results show
that PC neurons in the PEMS migrate circumferentially, cross the midline and
then turn radially at specific locations.
Morphological maturation and formation of nuclei
We examined these cells at a later stage, E18.5 to study the fate of the
EYFP-labelled cells. Fewer EYFP-labelled cells were visible in the PEMS at
this stage (data not shown). Conversely, those in the ECN and LRN regions
increased in number and occupied a wider area than was observed at E15.5
(Fig. 3A-C; n=10).
Many of the labelled cells in the deeper regions of these nuclei had
multipolar shapes with several processes that resembled dendrites (the left
panels of Fig. 3D-F), implying
that lRL-derived ECN and LRN precursors are nearly mature as they migrate deep
into the medulla.
|
;
Walberg, 1952;
Kapogianis et al., 1982). Most
of these cells in the dorsal cluster and those in the ventral medulla
expressed Mbh2 (the right panels of
Fig. 3D-G). Although labelled
cells were also found ipsilaterally, in regions corresponding to the ECN and
LRN, there were only a few of these cells, and most were Mbh2
negative (data not shown). These results show that lRL-derived cells generated
from E12.5 onwards give rise to ECN and LRN on the contralateral side.
Ectopic formation of ECN and LRN by introduction of a dominant negative form of cadherins
Migrating ECN/LRN neurons show a transition in their direction of migration
at a specific point along their migratory pathway, raising the possibility
that these neurons may recognize cue(s) for the transition. A question then
arises of why they do not show a transition on the ipsilateral side. As we
discussed above, it seems unlikely that this is due to underdeveloped
environmental cues (Fig.
2D).
An alternative possibility is that these neurons acquire competence to respond such presumptive cues depending on the time after their generation. As a tool to test this possibility, we used a dominant-negative form of cadherins [EGFP-Ncad(t)], which slows down migration of PC neurons (H.T., D.K., K. Nishida and F.M., unpublished). In E18.5 control preparations, most strong Venus signals were observed in the contralateral ECN and LRN region (Fig. 4A,B). By contrast, when EGFP-Ncad(t) was co-electroporated with Venus, a large number of EGFP-Ncad(t)-transfected neurons appeared to form aggregates in ECN and LRN regions on the ipsilateral side (Fig. 4C,D; n=9/12). In transverse sections, we found that many Venus-labelled cells formed clusters in deeper regions of ECN and LRN (Fig. 4E,F; data not shown), intermingled with Mbh2 signals that may presumably represent the neurons of contralateral origin (insets in Fig. 4E). These observations suggest the possibility that the neurons whose migration was delayed by transfection of EGFP-Ncad(t) could recognize the presumptive cues for transition to radial direction on the ipsilateral side and are consistent with the interpretation that ECN and LRN neurons normally acquire responsiveness to the cues depending on an intrinsic, age-dependent mechanism. Moreover, these results indicate that midline crossing of ECN and LRN neurons is not prerequisite for nucleogenesis by these neurons.
PGN and RTN neurons undergo partial midline crossing and radial migration
Although the midline crossing of PGN and RTN neurons has not been described
previously, we did observe midline crossing of EYFP-labelled cells.
Fig. 5A shows EYFP-labelled
cells in the pons of an E14.5 embryo. A substantial number of EYFP-labelled
cells had apparently migrated across the ventral midline (broken line)
(n=12). At E15.5, cells that appeared to have crossed the midline
were dispersed dorsally (Fig.
5B, n=12).
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At E18.5, EYFP-labelled cells formed bilateral clusters that appeared to correspond to the PGN and the RTN. Rostrally, EYFP-labelled cells appeared to occupy the entire territory of the ipsilateral PGN (Fig. 6A,E, n=10). However, they were largely confined to the dorsal aspect in the contralateral PGN (Fig. 6A), and axonal profiles, perhaps from the ipsilateral PGN neurons, were found (arrowheads in Fig. 6F) in the ventral pons. Caudally, at the level of the RTN, radially migrating EYFP-labelled neurons were nearly symmetrically distributed on both sides (Fig. 6C, n=10). Such ventricle-directed neurons were found in the PGN and RTN, and these cells had several branched processes (Fig. 6G-J) that appeared to be immature dendrites. These results indicate that nucleogenesis by EYFP-labelled cells takes place in the PGN and RTN regions on both sides of the neural tube. EYFP-labelled neurons largely coincided with Mbh2 (Fig. 6A-D,G-J), further supporting the conclusion that these bilateral clusters of EYFP-labelled cells are PGN and RTN neurons.
Birthdate-dependent midline crossing of PGN/RTN neurons
The PGN neurons that crossed the midline were found in the dorsal PGN
(Fig. 6A,C). Because
earlier-generated PGN neurons are located in deeper regions of the hindbrain
(Altman and Bayer, 1987b;
Altman and Bayer, 1997), it is
possible that only the early-born PGN neurons can cross the midline. To test
this hypothesis, BrdU was injected at E12.5, E13.5 and E14.5, into pregnant
dams that had been electroporated with EYFP at E12.5
(Fig. 7). At E18.5, about
one-third of the EYFP-positive neurons on the contralateral side were BrdU
positive in embryos injected with BrdU at E12.5
(Fig. 7A,C). However, only a
few EYFP/BrdU double-positive neurons were observed following BrdU injection
at E14.5 (Fig. 7B,D). Most
E14.5-born RTN neurons did not express EYFP on the contralateral side
(Fig. 7B). Counts of
BrdU-labelled cells showed that less than 3% of the EYFP-positive neurons
generated at E14.5 reached the contralateral side, compared with more than 30%
for the neurons born at E12.5 (Fig.
7E). Therefore, it is the earlier (before E14.5)-generated PGN and
RTN neurons that cross the midline.
Radial fibres may serve as a substrate for ventricle-directed migration of PC neurons
As pial surface-directed radial migration occurs along the radial fibres in
the cerebral cortex (Rakic,
1990), we next investigated whether the ventricle-directed
migration of PC neurons also takes place along radial fibres. At E15.5, some
EYFP-labelled cells in the myelencephalon became ventricle oriented
(Fig. 8A,C). The radially
extending fibres that span the wall of the hindbrain were immunopositive for
nestin, a marker for radial glia-like neuroepithelial cells
(Lendahl et al., 1990;
Mignone et al., 2004)
(Fig. 8C, middle panel). The
leading processes of ventricle-oriented ECN and LRN neurons were closely
apposed to nestin-positive fibres (data not shown and
Fig. 8C, right; n=10
cells in 10 slices from three embryos for ECN, n=9 cells in four
slices from two embryos for LRN). Ventricle-directed PGN and RTN neurons in
the ventral metencephalon were also closely associated with nestin-positive
radial fibres (Fig. 8B,D)
(n=14 cells in three slices from two embryos).
Electron microscopic observations of the pontine region confirmed that
cells that appear to be PC neurons directly contacted radially aligned fibres.
At a low magnification, radial fibres could be readily recognized by their
long and radial extension and their termination at the pial surface
(Fig. 8E,F and data not shown).
At a high magnification they were characterized by the presence of glycogen
particles (arrows in Fig. 8F)
(Gadisseux et al., 1990).
Presumptive PC neurons could also be recognized by their relatively dark and
ribosome-rich cytoplasm (Fig.
8E,F) (Ono and Kawamura,
1990). Consistent with light microscopic observations described
above, such cells were oriented both tangentially and towards the ventricle.
We examined whether PC cells make contact with radial fibres. As illustrated
in Fig. 8F, we occasionally
observed a contact between radial fibres and cell bodies of presumptive PC
neurons. Contacts between ribosome-rich thin processes which appear to be the
leading or trailing process of PC neurons and radial fibres were also
encountered (data not shown). Altogether, these results strongly support the
conclusion that nestin-positive radial fibres in the hindbrain mediate the
ventricle-directed radial migration of PC neurons.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Identification of EYFP-labelled cells
Our observations that Mbh2 is expressed by EYFP-positive cells
strongly suggest that these cells are PC neurons. Mbh2 is expressed
in the rhombic lip early in development
(Bulfone et al., 2000), and its
expression pattern corresponds to the anatomically defined positions of
mossy-fibre-projecting PC neurons (Li et
al., 2004). The axons of EYFP-positive cells, in our embryos
examined at E16.5 and later, projected to the cerebellum (D.K., T.S. and F.M.,
unpublished), which further strengthens the hypothesis that the EYFP-labelled
cells are future PC neurons.
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;
Altman and Bayer, 1997). This
may be largely because the neurons in these nuclei are generated before E12.5
(Taber-Pierce, 1973), and have
left the lRL by the time of our electroporation, although there is the
possibility that inferior olivary neurons have a distinct origin
(Wang et al., 2005;
Landsberg et al., 2005).
Midline crossing of future PC neurons
Previous in vitro studies that labelled presumptive ECN/LRN cells with DiI
injections (Kyriakopoulou et al.,
2002) or EGFP electroporation
(Taniguchi et al., 2002)
demonstrated migration across the midline. In this study, we have corroborated
these findings by providing in vivo evidence that future ECN and LRN neurons
originate from the contralateral lRL. Consistent with our results, a recent
analysis of Rig1-deficient mice implied midline crossing of these
neurons (Marillat et al.,
2004). An unexpected finding was that a subset of PGN and RTN
neurons have a contralateral origin (Figs
5,
6).
Possible mechanisms involved in tangential migration of PC neurons
There are several important phases in the nucleogenesis of PC neurons.
LRL-derived neurons must first determine which of the two migratory pathways
they should take (Fig. 1). A
recent study demonstrated that Mbh1/Barhl2, a homolog of
Mbh2 (Saito et al.,
1998), is expressed in AEMS, but not in PEMS
(Mo et al., 2004). It is
therefore possible that lRL-derived neurons use cues under the control of
Mbh1 when choosing one of these migratory pathways.
We have found that tangential migration of PC neurons is terminated by the
transition to ventricle-directed migration, not merely by ceasing to migrate
along their tangential route. The transition and turning could be regulated
either by an intrinsic or an extrinsic mechanism. The former is consistent
with a previous observation of facial branchiomotor neurons, which regulate
expression of surface molecules depending on migration phase
(Garel et al., 2000) (for a
review, see Chandorasekhar, 2004). However, the fact that the transitions in
our study occurred at restricted sites in a protracted period of development
during which the size of the neural tube is dramatically increased favours the
environmental cue mechanism. Moreover, our results that introduction of
EGFP-Ncad(t) caused formation of ECN and LRN ipsilaterally but in
appropriate locations support the idea that environmental cues exist that
induce radial turning at defined locations along the circumferential axis. An
interesting possibility is that the transition involves radial fibres at
transition points with distinct properties that induce changes in the
migratory behaviour of the PC neurons.
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Radial-fibre associated, ventricle-directed radial migration
After the phase of tangential migration, PC neurons start migrating towards
the ventricle. The role of radial glial cells as a substrate for pial
surface-directed migration of cortical neurons is well documented
(Marin and Rubenstein, 2003;
Rakic, 1990;
Hatten and Mason, 1990). Their
role in ventricle-directed migration, however, is poorly understood. Here,
close association of EYFP-labelled neurons with nestin-positive fibres under
the light microscope observed here suggests that PC neurons use radial fibres
as a substrate for ventricle-directed migration. This view was strongly
supported by our electron microscopic observations, which demonstrated a
direct contact of a migrating cell with radial fibres
(Fig. 8). Our results provide
the first compelling evidence that nestin-positive radial fibres may guide
embryonic neurons towards the ventricle, which is essential for the formation
of nuclei in appropriate positions.
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Molecular mechanisms for the ventricle-directed radial migration may be
distinct from those for pial-surface directed migration, because genes that
play crucial roles in pial-surface directed migration of cortical neurons such
as cdk5/p35 (Ohshima et al.,
1996; Gupta et al.,
2003; Hatanaka et al.,
2004) and reelin (Ogawa et
al., 1995) do not appear to affect the nucleogenesis of
mossy-fibre-projecting PC neurons (Ohshima
et al., 2002).
Specification of distinct neuronal cell types in the hindbrain
The mossy-fibre-projecting PC neurons are characterized by an early
(E9.5-11.5) expression of some transcription factors
(Li et al., 2004;
Rodriguez and Dymecki, 2000;
Wang et al., 2005), before
they leave the lRL, suggesting that they are already specified before they
start migration. However, they have distinct migratory pathways and modes of
migration, and their eventual positions are dispersed in the hindbrain
(Rodriguez and Dymecki, 2000;
Wang et al., 2005) (this
study). These features cannot be explained simply by the expression of such
genes. There must be some mechanism that allows each subset of PC neurons to
express different transcription factors that control the responsiveness of
these neurons to environmental cues. To fully understand the molecular
mechanisms involved in the development of distinct cell types of PC neurons at
specific locations in the hindbrain, the factors that may be involved in the
regulation of PC neuron migration remain to be investigated. Our results may
help in unravelling the mechanisms underlying these events.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Cold Spring Harbor Laboratory, Beckman 220, One Bungtown
Road, Cold Spring Harbor, NY 11724, USA
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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