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First published online 3 October 2007
doi: 10.1242/dev.005447
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1 Institut für Anatomie und Zellbiologie, Albert-Ludwigs-Universität
Freiburg, D-79104 Freiburg, Germany.
2 Neurologische Universitätsklinik, Neurozentrum,
Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.
3 Max-Planck-Institut für Immunbiologie, D-79108 Freiburg, Germany.
4 Zentrum für Neurowissenschaften, Albert-Ludwigs-Universität
Freiburg, D-79104 Freiburg, Germany.
Author for correspondence (e-mail:
Michael.Frotscher{at}anat.uni-freiburg.de)
Accepted 16 August 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Lipoprotein receptors, disabled 1, Reelin, Layer formation, Neuronal migration
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Berry and Rogers, 1965;
Rakic and Caviness, 1995;
Takahashi et al., 1999). The
formation of individual neuronal layers involves migration of neurons in
radial and tangential directions to their final laminar positions. To generate
an inside-out pattern, cells migrating to the cortical plate must be able to
pass earlier generated neurons and stop at their appropriate destinations. The
nature of interaction of migrating cells with their environment and the
molecular signals that regulate this process and subsequent differentiation
are complex, and as yet poorly understood (reviewed by
Marin and Rubenstein,
2003).
One pathway regulating the migration of neurons involves the extracellular
matrix protein reelin (reviewed by Rakic
and Caviness, 1995; Curran and
d'Arcangelo, 1998; Frotscher,
1998; Tissir and Goffinet,
2003; Soriano and Del Rio,
2005; Förster et al.,
2006a; Förster et al.,
2006b). Reelin is a glycoprotein secreted by Cajal-Retzius cells
in the developing marginal zone of the cortex and other laminated brain
regions (D'Arcangelo et al.,
1995; D'Arcangelo et al.,
1997; Lambert de Rouvroit and
Goffinet, 1998; Meyer and
Goffinet, 1998; Rice et al.,
2001). Different models of reelin action have been proposed.
Reelin may act as a stop signal (Dulabon
et al., 2000), a chemoattractant
(Gilmore and Herrup, 2000) and
a detachment signal for migrating neurons
(Hack et al., 2002;
Sanada et al., 2004).
Neurons migrating radially towards the cortical plate express at least
three essential elements of the reelin signaling cascade: the two reelin
receptors Vldlr (very low density lipoprotein receptor) and ApoER2
(apolipoprotein E receptor 2; also known as Lrp8 - Mouse Genome Informatics)
(D'Arcangelo et al., 1999;
Trommsdorff et al., 1999;
Hiesberger et al., 1999), and
the adapter molecule disabled 1 (Dab1) that binds to the intracellular domains
of these receptors (Howell et al.,
1997; Sheldon et al.,
1997; Ware et al.,
1997; Rice et al.,
1998; Trommsdorff et al.,
1999). Binding of reelin to its membrane receptors VLDLR or ApoER2
has been shown to induce phosphorylation of Dab1
(Hiesberger et al., 1999;
Howell et al., 1999;
Trommsdorff et al., 1999).
Mutations in the reelin gene, the Dab1 gene and in both the Vldlr and ApoER2
gene result in a reeler-like phenotype characterized by a severely altered
cortical layering (Howell et al.,
1997; Sheldon et al.,
1997; Ware et al.,
1997; Lambert de Rouvroit and
Goffinet, 1998; Trommsdorff et
al., 1999). The analysis of single receptor mutants, however,
revealed a minor phenotype with Vldlr being more important for the development
of the cerebellum and ApoER2 for cortical lamination
(Trommsdorff et al., 1999;
Benhayon et al., 2003). To
further characterize possible individual roles of these two reelin receptor
molecules for the development of cortical layers, we have examined cortical
lamination in Vldlr and ApoER2 single and double-knockout mutants, reeler mice
and Dab1 mutants using layer-specific markers and bromodeoxyuridine (BrdU)
labeling. With this combined approach we provide new evidence for divergent
roles of ApoER2 and Vldlr in neuronal migration and cortical lamination.
Whereas ApoER2 is important for the proper migration of late generated
neurons, Vldlr mediates a stop signal for reelin, preventing migrating neurons
from entering the marginal zone.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In situ hybridization
Probe generation
Total RNA from P0 wild-type brains was reverse-transcribed to cDNA with
reverse transcriptase (Supercript II, Invitrogen) and used as a template for
the PCR reaction (Eppendorf, Hamburg, Germany). The following oligonucleotide
primers were used (MWG-Biotech AG, Ebersberg, Germany): ApoER2
forward 5'-GCTGTCATTGGGGTCATCG-3', reverse:
5'-GCTTGCACTTGACGACAGGC-3'. The primers also included T7
polymerase binding sites (not listed). The combination of primers led to the
amplification of the expected single band of 398 bp. The PCR template was
purified (Qiaquick purification kit, Qiagen, Hilden, Germany), sequenced
(GATC, Konstanz, Germany), and used for in vitro transcription.
In vitro transcription
In vitro transcription was performed with 1 µg linearized plasmid DNA or
PCR template, in the presence of ATP, GTP, CTP and digoxigenin-11-UTP (DIG),
RNasin, transcription buffer and T3, SP6 or T7 RNA polymerase (Roche
Diagnostics), for 2 hours at 37°C following the manufacturer's
recommendations.
RNA detection by in situ hybridization
For in situ hybridization the following DIG-labeled riboprobes were used:
Cux2 (Zimmer et al.,
2004) (also known as Cutl2 - Mouse Genome Informatics),
ER81 (Lin et al.,
1998) (also known as Etv1 - Mouse Genome Informatics),
RORbeta (Schaeren-Wiemers et al.,
1997) (also known as Rorb - Mouse Genome Informatics),
Tle4 (Beffert et al.,
2006) and ApoER2. Brains (n=5-10 for each marker
and developmental time point) were fixed in freshly prepared 4% PFA in
1x PBS, cryoprotected in 30% sucrose, and frozen in isopentane at
-60°C. In situ hybridization was performed on 50 µm free-floating
cryostat sections as described previously
(Haas et al., 2000). Briefly,
free-floating sections were prehybridized in hybridization buffer (50%
formamide, 4x SSC, 50 mM NaH2PO4, 250 µg/ml
heat-denatured salmon sperm DNA, 100 µg/ml tRNA, 5% dextransulfate and 1%
Denhard's solution) for 60 minutes at 45°C or 65°C. Hybridization was
performed in the same buffer including 700 ng/ml of the riboprobes at 45°C
or 65°C overnight. After hybridization, the sections were washed in
2x SSC (twice for 15 minutes each) at room temperature, 2x SSC and
50% formamide, 0.1x SSC and 50% formamide for 15 minutes each and
finally in 0.1x SSC (twice for 15 minutes each) at 55°C or 64°C.
Immunological detection with anti-DIG-AP was performed as recommended by the
manufacturer (Roche Diagnostics). Colorimetric detection was accomplished
using nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate
(BCIP).
For double fluorescent in situ hybridization (FISH), sections were
co-hybridized with a digoxigenin (DIG)-labeled ApoER2 or
RORbeta probe and a FITC-labeled Cux2 probe and processed as
described by Dufour et al. (Dufour et al.,
2006). To allow for a comparison of the layer-specific markers
with Nissl-stained sections, part of the sections were stained with Cresyl
Violet, dehydrated in ethanol and xylene, and coverslips applied with Histokit
(Shandon, Pittsburgh, USA).
Immunohistochemistry
Staining procedure Brains (n=4-10 for each genotype and
developmental time point) were fixed in 4% PFA in 1x PBS and sliced
sagittally on a vibratome (50 µm). Sections were pre-incubated for 60
minutes in blocking solution [10% fetal calf serum (FCS) in 1x PBS] at
room temperature. Subsequently, sections were incubated with the primary
antibodies in 3% FCS containing 0.1% Triton-X 100 in 1x PBS overnight at
4°C. After washing three times for 10 minutes each in 1x PBS at room
temperature, sections were incubated in secondary antibodies for 2 hours at
room temperature. After rinsing in 1x PBS (three times for 30 minutes),
sections were mounted in fluorescent mounting medium (DAKO).
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). Visualization of antibody binding by diaminobenzidine (DAB) staining was performed using the ABC Standard Kit (Vector Laboratories, Burlingame, USA) with DAB and H2O2 as substrates in accordance with the manufacturer's suggestions. Finally, sections were mounted on gelatin-coated slides, dehydrated and coverslips applied using Histokit.
Antibodies
The following primary antibodies were used: mouse anti-reelin (mAb5364,
1:250, Chemicon, Hofheim, Germany), mouse anti-NeuN (also known as Neuna60 -
Mouse Genome Informatics; mAb377, neuron-specific nuclear protein, 1:1000;
Chemicon), rabbit anti-calbindin (CB38, 1:1000; Swant, Bellinzona,
Switzerland) and rabbit anti-calretinin (769914, 1:1000; Swant), mouse
anti-GAD67 (also known as Gad1 - Mouse Genome Informatics; mAb5406, 1:1000;
Chemicon), rabbit anti-parvalbumin (PV28, 1:1000; Swant), guinea pig
anti-GLAST (also known as Slc1a3 - Mouse Genome Informatics; mAb1782, 1:500;
Chemicon), rabbit anti-brain lipid binding protein (BLBP; (also known as Fabp7
- Mouse Genome Informatics; AB9558, 1:500; Chemicon), mouse anti-nestin
(Rat401, 1:50; Developmental Studies Hybridoma Bank at the University of Iowa,
Iowa City, IA), rabbit anti-Tbr1 (AB9616, 1:10,000; Chemicon), goat anti-Foxp2
(N16) (sc-21069, 1:500; Santa Cruz Biotechnology, Heidelberg, Germany), and
mouse anti-SMI32 (1:100; Sternberger Monoclonals, Lutherville, Maryland, USA).
Secondary antibodies: goat anti-mouse Alexa Fluor 568 (A-11004, 1:1000;
Invitrogen), goat anti-rabbit Alexa Fluor 488 (A-11008, 1:1000; Invitrogen),
rabbit anti-goat Alexa Fluor 488 (A-11078, 1:300; Invitrogen) and biotinylated
horse anti-mouse (BA-2000, 1:250; Vector Laboratories).
Quantitative stereology
The number of NeuN-positive neurons was obtained by the optical
dissector/fractionator method (West et
al., 1991). For quantitative stereology, frontal sections of adult
brains (n=4 for each group) were visualized on a computer screen
attached to an Olympus BX60 microscope F5 (Olympus Optical, Düsseldorf,
Germany). A computer-controlled stepper motor stage and focus assembly allowed
movement in the x-, y- and z-axes. Cell counts were
performed using Stereo Investigator software (version 3.0; MicroBrightField,
Inc., Colchester, VT). The marginal zone, as the region of interest, was first
marked for every single section using low-power magnification (4x/0.10
objective). For subsequent cell counts, the following parameters were added to
the program: counting frame, 50x30 µm; guard zone, 2 µm, and
counting depth, 8 µm. Thereafter, using high-power magnification (oil
objective lens, 100x/1.35), NeuN-positive cells that fulfilled the
criteria of the unbiased counting rules were marked and added to the probe run
list. The total cell numbers estimated by the optical dissector were
subsequently analyzed by Wilcoxon's exact rank sum test.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To
investigate the role of these two reelin receptors in more detail, we first
used layer-specific markers to examine the laminar structure of the cerebral
cortex in ApoER2-/- and Vldlr-/- mice
at different developmental stages. The transcription factor ER81 is strongly
expressed by cells of cortical layer V
(Hevner et al., 2003).
Accordingly, we used ER81 as a marker for cortical cells committed to a deep
layer fate. These cells are born at early stages of cortical development,
around E12-E13 (Takahashi et al.,
1999). ER81-positive neurons are positioned in the inner
part of the cortex of wild-type mice 1 week after birth
(Fig. 1A). In general, the
pattern of ER81 staining was similar when we analyzed Vldlr
mutant mice: ER81-positive neurons were localized in an organized
layer in the inner part of the cortex (Fig.
1D). By contrast, in situ hybridization for ER81 in
ApoER2-/- mice showed positive cells in an organized layer
located ectopically in more superficial positions of the cortex
(Fig. 1G). This part of the
cortex is usually occupied by layer II-IV neurons. Since ER81
expression may not be restricted to layer V pyramidal cells
(Wonders and Anderson, 2005),
we studied additional markers of deep layer neurons, such as Tbr1,
Foxp2 and Tle4. Similar observations as with ER81 were
made when these markers were used (Fig.
2A-D).
The homeodomain transcription factor Cux2 is specifically expressed within
cortical layers II-IV (Zimmer et al.,
2004). Accordingly, the Cux2-positive population in
wild-type animals was positioned in the outer portion of the cortical plate,
which gives rise to the upper cortical layers II-IV
(Fig. 1B). In Vldlr
mutant mice, the Cux2-positive cells were also localized in the upper
part of the cortex (Fig. 1E)
but, unlike in wild-type mice, we observed numerous Cux2-positive
cells in layer I of the cortex (see also
Fig. 7). Furthermore, a sharply
separated marginal zone could not be discerned in Vldlr mutant mice
(compare Fig. 1B and E,
Fig. 7A and B). These findings
suggest an ectopic invasion of Cux2-positive neurons into the
cortical marginal zone. The invasion of layer I first became obvious at P7,
when this layer was large enough to be clearly defined.
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In order to compare the expression patterns of Cux2 and ER81 in ApoER2-/- mice and Vldlr-/- mice with those in other mutants lacking various components of the reelin signaling pathway, we performed in situ hybridization studies in reeler mice, Dab1-/- mice and ApoER2 Vldlr double-knockout mice using ER81 and Cux2 riboprobes. As expected, no distinct layer formation was visible in the three mutants, reflecting the more severe phenotype in these animals (see Fig. S1 in the supplementary material).
ApoER2 is important during late stages of cortical layer formation
In situ hybridization for Cux2 on sections of ApoER2
mutant mice revealed two bands of expression in the cortex, one in normal
superficial position, and a second one in an abnormal position deep in the
cortex (compare Fig. 1B with
H). Comparing the expression patterns of ER81 and
Cux2, the Cux2-positive upper band was localized directly
above the ER81-positive layer V
(Fig. 1G,H), suggesting that
these Cux2-labeled cells mainly belonged to layer IV. If this were
true, the deep Cux2-positive cells would mainly include neurons
normally destined to layers II-III. To confirm that the Cux2-positive
cells in the superficial band of the cortex of ApoER2 mutants were
relatively early generated neurons, we used BrdU labeling at different
developmental time points.
Following BrdU injections at E12.5 and E13.5, wild-type animals exhibited the most heavily BrdU-labeled cells in inner and middle locations of the cortex when analyzed at P0 (Fig. 3A,B). BrdU injections at E14.5-E16.5 mainly labeled cells destined to outer portions of the cortex (Fig. 3C-E). An altered pattern was observed in ApoER2 mutants (Fig. 3F-J). Here, BrdU-labeling at E13.5, and more clearly at E14.5 and E15.5, led to the formation of two separate BrdU-positive bands. Injection of BrdU at E16.5 labeled a distinct band of deeply located neurons in ApoER2 mutants (Fig. 3J). Together, these findings are in line with the observation of two separate layers of late born, Cux2-positive cells. They suggest that in ApoER2 mutants, a large fraction of late born neurons fail to migrate to their destinations in outer cortical layers.
To substantiate further that the upper BrdU-positive band in the
ApoER2 mutants represents neurons that are normally destined to layer
IV, we performed in situ hybridization for RORbeta, a marker for
layer IV neurons (Nakagawa and O'Leary,
2003). In adult wild-type animals, RORbeta mRNA
expression is, in fact, mainly seen in layer IV cells
(Fig. 4A). By contrast, in
ApoER2 mutants a dispersion of RORbeta-positive cells, with
strongest expression levels underneath the marginal zone and close to the
ventricle, is observed (Fig.
4E). Double in situ hybridization for RORbeta and
Cux2 in wild-type animals (Fig.
4B-D) revealed a Cux2-positive band just above
RORbeta-stained cells, but also double-labeled neurons
(Fig. 4D). By contrast, in
ApoER2 mutants Cux2-positive and RORbeta-positive
neurons were scattered, forming cell accumulations underneath the marginal
zone and near the ventricular zone (Fig.
4F-H). We conclude that early born layer IV neurons migrate
properly, consistent with the superficial RORbeta-positive
population, whereas later generated layer IV and layer II-III cells remain
close to the proliferative zone. These data together with the BrdU studies
suggest that ApoER2-mediated signaling is important for late phases of
neuronal migration in the cortex.
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Recent studies indicated that correct layer destination of cortical
GABAergic interneurons does not directly depend on reelin signaling
(Pla et al., 2006).
Interneurons seem to invade their target layers well after synchronously
generated projection neurons reach their final destinations, so that
projection neurons guide cortical interneurons to their appropriate layer.
Since we found a prominent migration defect in ApoER2 mutants, we
expected also interneurons to be misplaced in these animals. Immunostaining
for the interneuron marker calretinin revealed positive neurons mainly in
superficial cortical layers in wild-type animals (see Fig. S2A in the
supplementary material). As expected, in ApoER2 mutants
calretinin-positive cells were found in superficial and deep cortical layers
(see Fig. S2B in the supplementary material). Immunostaining for parvalbumin,
another marker of GABAergic interneurons, shows a scattered distribution of
labeled neurons in sections of wild-type animals and ApoER2 mutants
(see Fig. S2C,D in the supplementary material).
The radial glial scaffold is not altered in ApoER2 mutants
Our results indicate that ApoER2 is involved in the migration of late
generated neurons that mainly use radial glia-guided migration to reach their
destinations in superficial cortical layers
(Nadarajah and Parnavelas,
2002). Could the migration defect seen in ApoER2 mutants
be due to an altered radial glial scaffold? We studied the organization of the
radial glial scaffold at E16.5 by using antibodies against BLBP, nestin and
GLAST, but were unable to find obvious differences in morphology and
arrangement of radial glial cells when comparing sections of wild-type and
mutant cortex (Fig. 6A-F). As
the glial scaffold does not seem to be altered in the mutant cortex, the
observed neuronal phenotype in ApoER2-/- mice could be due
to a failure of the neurons to attach properly to the radial glial fiber. This
attractive hypothesis needs to be tested in future real-time microscopy
studies.
Cortical neurons invade the marginal zone in Vldlr mutants
In situ hybridization for Cux2 showed an invasion of cells into
the marginal zone of Vldlr mutants
(Fig. 7A,B), but not of
ApoER2 mutants, suggesting different roles of these two lipoprotein
receptors. As Cux2 expression only indicates a neuronal subpopulation
within layers II-IV, we performed immunohistochemistry for the neuronal marker
NeuN. In fact, staining for NeuN revealed more prominent differences when
comparing the presence of neurons in the marginal zone of wild-type mice
(Fig. 7C),
ApoER2-/- (Fig.
7D) and Vldlr-/-
(Fig. 7E) mice. Stereological
estimation of the number of NeuN-positive cells in the marginal zone and
subsequent statistical analysis revealed a significant increase in
NeuN-positive neurons in Vldlr mutant mice compared to both wild-type
animals and ApoER2 mutants. By contrast, no significant difference
was found when wild-type animals and ApoER2 mutants were compared
(Fig. 7F).
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). One
subpopulation consists of cortical interneurons born in the subpallium,
migrating tangentially to the cortex. The other population comprises cortical
projection neurons born in the dorsal telencephalon. These cells migrate
radially from the subventricular zone (SVZ) to superficial positions before
they differentiate into upper cortical layer neurons
(Tan and Breen, 1993;
Tan et al., 1998;
Nadarajah and Parnavelas,
2002). To determine the types of cells in the marginal zone of
Vldlr-/- mice, we used antibodies against different
interneuron markers such as GAD67, parvalbumin and calretinin, and antibodies
against calbindin and reelin and the pyramidal neuron markers SMI32 and Tbr1
(Fig. 8). Immunostaining for
calbindin, a marker of both interneurons and pyramidal cells, revealed many
cells in the marginal zones of Vldlr mutants when compared with
wild-type animals (Fig. 8A,B).
Similarly, we found SMI32-positive pyramidal cells and Tbr1-positive neurons
only in the marginal zones of Vldlr mutant mice
(Fig. 8C-G). As known from
studies in the reeler mutant, some of these displaced pyramidal cells were
inverted with the apical dendrite directed towards the white matter
(Fig. 8E-G). We were unable to
find clear-cut differences in the labeling of interneurons in layer I between
Vldlr mutants and wild-type animals using antibodies against
calretinin, parvalbumin, GAD67 and reelin (see Fig. S2E-L in the supplementary
material). The different roles of ApoER2 and Vldlr do not seem to be restricted to the migration of neocortical neurons. Thus, we observed a neuronal migration defect in the olfactory bulb of ApoER2 mutants but not Vldlr mutants when staining sections of adult animals for calbindin (see Fig. S3A-C in the supplementary material). As in the neocortex, no alterations of the radial glial scaffold were observed (see Fig. S3D,E in the supplementary material).
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Frotscher, 1998).
ApoER2 mutants, but not Vldlr-/- mice, show
migration defects of late generated neurons destined for superficial
layers.
|
|
;
Nadarajah and Parnavelas,
2002). Early generated neurons, giving rise to the formation of
deep cortical layers and the establishment of corticofugal connections, are
likely to use somal translocation to move from the germinal ventricular zone
to their definitive positions in the cortical plate. This mode of migration
may be necessary in the absence of a glial scaffold early in the development
of the cortex. Later on in development, when a radial glial scaffold has
formed (Kriegstein and Götz,
2003), neurons destined to superficial cortical layers use radial
glial processes as a template to move away from the proliferative zone. A
glia-guided migration appears necessary to govern the increasingly longer
migration of late generated neurons. This way, the layers of the cortex are
formed in an inside-out manner (Caviness
and Rakic, 1978; Rakic and
Caviness, 1995).
The cortex of the reeler mutant is characterized by an abnormal cortical
lamination despite a virtually normal development of the preplate
(Caviness, 1982;
Sheppard and Pearlman, 1997).
A plausible explanation may be that early generated cortical neurons adopt a
mode of migration that is unaffected by the cascade of signaling mechanisms
that regulate the glia-guided migration of late generated cortical neurons.
The data of the present study are consistent with this hypothesis. In the
ApoER2 mutant, early generated layers are formed almost normally,
with one subpopulation of layer IV neurons lying above layer V. These cells
are labeled with the layer IV-specific marker RORbeta. Thus, the
formation of these early layers is largely independent of ApoER2 signaling. By
contrast, the formation of superficial, late generated layers is severely
altered. Late born cells are unable to bypass earlier ones and remain close to
the ventricular zone. It is well-known that the cells destined to layers IV
and V are among early generated neurons that start to migrate before E14.5,
likely using somal translocation as their mode of migration. By contrast,
later generated neurons follow thereafter and may increasingly use radial
glia-guided migration (Nadarajah and
Parnavelas, 2002; Sanada et
al., 2004). As these late forming layers are affected in the
ApoER2 mutant, we suggest that late glia-guided migration is
controlled by ApoER2 signaling. This is consistent with the finding that in
the wild-type cortex strongest ApoER2 mRNA expression was found in
upper cortical layers. Since we were unable to find obvious changes in the
radial glial scaffold, we assume that the interaction of the migrating neuron
with the radial fiber is altered in a yet unknown way. Studies are in progress
analyzing glia-guided migration of late generated cortical neurons in
wild-type mice and ApoER2 mutants by means of real-time
microscopy.
Vldlr - a stop signal for migrating cortical neurons?
In situ hybridization for Cux2 and immunostaining for NeuN and
subsequent stereological quantification revealed that significantly more
neurons were present in the marginal zone of Vldlr mutants than in
wild-type animals or ApoER2 mutant mice. In the marginal zone of
adult wild-type animals some GABAergic interneurons but no pyramidal cells are
present. By contrast, staining with SMI32 and Tbr1 showed a small population
of pyramidal cells in the marginal zone of Vldlr mutant mice that was
absent in wild-type animals and ApoER2 mutants. The presence of these
neurons in the marginal zone of Vldlr mutants indicates that at least
some of these cells are early-generated pyramidal cells that were not
prevented from invading the marginal zone.
During development, Vldlr is expressed in radially migrating neurons that
are about to signal reelin in the marginal zone
(Trommsdorff et al., 1999;
Perez-Garcia et al., 2004).
This is consistent with a role of Vldlr as a receptor terminating the
migratory process. Signaling via Vldlr results in the phosphorylation of Dab1
at tyrosine 220 and 232, which in turn leads to the detachment of the
migrating neuron from the radial glial fiber
(Sanada et al., 2004). Thus,
it appears that Vldlr is the receptor mediating the `stop signal' function of
reelin in the marginal zone.
Another scenario may hold true for GABAergic interneurons known to migrate
from the lateral ganglionic eminence in a tangential direction to settle in
the various layers of the cerebral cortex. In late stages of corticogenesis
(E14-E16), GABAergic interneurons are first destined to the ventricular zone
of the cortex where they stay for a short while before they migrate radially
towards the pial surface to their final destinations in the cortical plate
(Nadarajah et al., 2002).
Recently it has been shown that tangential migration and layer acquisition of
cortical GABAergic interneurons are independent of reelin signaling
(Pla et al., 2006); however,
it has been postulated that interneurons require a normal distribution of
projection neurons to adopt their correct laminar positions
(Pla et al., 2006).
In conclusion, consistent with the present findings, reelin, mediated via Vldlr is likely to provide a stop signal for radially migrating cells of the cortical plate, preventing them from entering the marginal zone. Together with the aberrant migration of late generated cortical neurons in ApoER2 mutants, the results of the present study point to specific functions of the two lipoprotein receptors for reelin.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/21/3883/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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