Reelin Together with ApoER2 Regulates Interneuron
Migration in the Olfactory Bulb
Sabine Hellwig1,2*, Iris Hack3, Birgit Zucker4, Bianka Brunne5, Dirk Junghans2*¤
1Department of Psychiatry and Psychotherapy, University of Freiburg Medical School, Freiburg, Germany, 2Institute of Anatomy and Cell Biology I, University of Freiburg,
Freiburg, Germany, 3Institute of Neuroscience and Medicine (INM-2), Research Center Ju ¨lich, Ju ¨lich, Germany, 4Department of Neurology, Neurocenter, University of
Freiburg Medical School, Freiburg, Germany, 5Institute of Structural Neurobiology, Center for Molecular Neurobiology, Hamburg, Germany
One pathway regulating the migration of neurons during development of the mammalian cortex involves the extracellular
matrix protein Reelin. Reelin and components of its signaling cascade, the lipoprotein receptors ApoER2 and Vldlr and the
intracellular adapter protein Dab1 are pivotal for a correct layer formation during corticogenesis. The olfactory bulb (OB) as
a phylogenetically old cortical region is known to be a prominent site of Reelin expression. Although some aspects of Reelin
function in the OB have been described, the influence of Reelin on OB layer formation has so far been poorly analyzed. Here
we studied animals deficient for either Reelin, Vldlr, ApoER2 or Dab1 as well as double-null mutants. We performed
organotypic migration assays, immunohistochemical marker analysis and BrdU incorporation studies to elucidate roles for
the different components of the Reelin signaling cascade in OB neuroblast migration and layer formation. We identified
ApoER2 as being the main receptor responsible for Reelin mediated detachment of neuroblasts and correct migration of
early generated interneurons within the OB, a prerequisite for correct OB lamination.
Citation: Hellwig S, Hack I, Zucker B, Brunne B, Junghans D (2012) Reelin Together with ApoER2 Regulates Interneuron Migration in the Olfactory Bulb. PLoS
ONE 7(11): e50646. doi:10.1371/journal.pone.0050646
Editor: Benjamin Arenkiel, Baylor College of Medicine, United States of America
Received July 1, 2012; Accepted October 23, 2012; Published November 29, 2012
Copyright: ? 2012 Hellwig et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Deutsche Forschungsgemeinschaft (www.dfg.de), grant numbers: Ha4320/1-1, SFB 592 and SFB 780. The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (SH); firstname.lastname@example.org (DJ)
¤ Current address: Max Planck Institute of Immunbiology and Epigenetics, Dep. of Molecular Embryology, Freiburg, Germany, and Institute of Embryology and
Stem Cell Biology, Dep. of Biomedicine, University of Basel, Basel, Switzerland
The OB is a phylogenetically old cortical region, which like the
neocortex is laminated and consists of five individual layers:
glomerular layer (GL), external plexiform layer (EPL), mitral cell
layer (MCL), internal plexiform layer (IPL) and granule cell layer
(GCL). These layers are formed in an inside-outside manner
during development . Distinct neuronal populations are found
in the layers of the OB: mitral cells as principal projection neurons
and in addition a morphologically similar but biochemically
heterogeneous group of interneurons [2,3,4,5]. Subsets of inter-
neurons, such as granule cells, periglomerular cells and inter-
neurons of the external plexiform layer, occupy distinct layers and
are therefore distinguishable based on their position within the OB
The OB is one of the few structures in the mammalian central
nervous system in which newly generated neurons are continu-
ously produced throughout adulthood [7,8]. At embryonic stage,
interneurons have been shown to arise from the lateral ganglionic
eminence and dorsal telencephalon [9,10,11,12,13,14] whereas
postnatally interneurons derive from the anterior part of the
subventricular zone (SVZ) of the lateral ventricle [15,16,17,18].
From this neurogenic region, precommitted neuroblasts migrate
tangentially in a chain-like organization into the OB, forming the
rostral migratory stream (RMS). After entering the OB, neuro-
blasts detach from the RMS, switch from chain to radial migration
and ascend radially into the defined layers where they finally
mature. The interaction of radial migrating cells with their
environment and the molecular signals underlying and regulating
this process are as yet poorly understood (reviewed in ).
One key signaling pathway known to orchestrate migration and
layer formation involves the extracellular matrix protein Reelin
(reviewed by [1,20,21,22,23,24]). Canonical Reelin signaling is
mediated by the two membrane bound lipoproteinreceptors
apolipoprotein E receptor 2 (ApoER2) and very low density
receptor (Vldlr) [25,26,27] and the cytoplasmatic adapter protein
disabled 1 (Dab1) [27,28,29,30,31]. Binding of Reelin to one of its
receptors induces phosphorylation of Dab1 essential to trigger
downstream signaling [26,27,32]. Null mutations in the Reelin or
Dab1 genes as well as Vldlr:ApoER2 double-null animals result in
reeler-like phenotypes characterized by severely altered cortical
layering [27,28,30,31,33]. Several modes of Reelin action have
been identified so far. Reelin can act as stop signal ,
chemoattractant  and in addition as detachment signal for
migrating neurons [36,37]. However, which of these functions are
responsible for correct cortical layering is heavily debated.
Reelin is prominently expressed in the OB  and abnormal-
ities in the OB of reeler mice have been described previously
including a size reduction as well as morphological alterations in
the periglomerular, mitral and granule cell layers [36,39]. Reelin
also acts as a detachment signal in the OB by inducing the switch
from tangential oriented chain-migration of RMS neuroblasts to
radial migration . However, studies on ApoER2, Vldlr, Dab1
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and Reelin deficient animals concluded that chain migration of
neuroblasts within the RMS and SVZ might be independent of
Reelin . Nevertheless, the precise role of Reelin signaling
components in OB layering and migration remains elusive.
In the present study we aimed to address this question.
Therefore, we examined the integrity of the RMS and the
detachment and lamination process in the OB of Vldlr2/2,
ApoER22/2single and double knockouts, Dab12/2and reeler
mutants by analyzing precursor migration in a matrigel explant
culture system, BrdU pulse labeling experiments and in addition
immunhistochemical analysis of OB layer formation and cell
positioning at different developmental time-points. In this context,
we demonstrate that ApoER2 is the main receptor mediating the
detachment signal of neuroblasts and correct migration of early
generated interneurons. The comparison of reeler and Dab1-
mutants also suggested that non-canonical Dab1-independent
Reelin signaling might play a role in OB layer formation.
Materials and Methods
Experiments were performed in agreement with the German
law on the use of laboratory animals and institutional guidelines of
the University of Freiburg. The use of animals was approved by
‘‘Regierungspra ¨sidium Freiburg (Freiburg regional council)’’ and
the animal welfare office of the University of Freiburg Ref. No. X-
08/04A. All animal experiments were performed at the University
of Freiburg, Dept. of Neuroanatomy.
Reeler mice were maintained on a B6/C3Fe background.
ApoER22/2, Vldlr2/2single and double mutant mice as well as
Dab12/2mutants were maintained on a mixed 129SvEv 6
C57BL/6J background. The day of birth was considered postnatal
day (P) 0.
RNA Preparation and RT-PCR
For cDNA synthesis total RNA was isolated from OBs of P0, P7,
P14 and adult wild-type mice and ApoER22/2mutant animals
(n=2–4 per genotype and developmental time point) using a tissue
homogenizer and Trizol reagent (Invitrogen). Total RNA was
DNase 1 treated (30 min, Roche Diagnostics) and purified
(RNeasy mini kit, Qiagen). cDNA synthesis was performed using
random primers (Promega) and Superscript II reverse transcrip-
tase (Invitrogen). Dilution series were made and PCR performed
with primers designed against b-actin for each individual cDNA to
equalize template cDNA concentrations. All PCRs were per-
formed by using junction primers specific for the individual
members of the Reelin signaling cascade (Fig. 1B). All samples
were tested on genomic and cDNA to confirm specificity and
purity of the template. Individual PCR reactions were run in
parallel and repeated at least three times.
Real time-PCR (Q-PCR) studies were performed with a Bio-
Rad iCycler by using SYBR-Green PCR Master Mix (Applied
Biosystems, Darmstadt, Germany) through 50 PCR cycles (95uC
for 30 s, 57uC for 60 s, 72uC for 90 s). Each cDNA sample was
run in triplicates for the target and the normalizing gene (ß-actin)
in the same 96-well plate. Specificity of amplicons was determined
by melt curve analysis and gel electrophoresis. Sequences of used
junction primers are listed in Fig. 1B.
Data analysis and statistics.
interest in each sample was calculated for Q-PCR by normaliza-
Expression of the mRNA of
tion of Ct values to the reference RNA (ß-actin) using the
equation: V=(1+E reference) (Ct reference)/(1+E target) (Ct
target) in order to correct for potential differences in PCR
amplification efficiencies. V=relative value of target gene
normalized to reference (ß-actin), E=PCR amplification efficien-
cy, Ct=threshold crossing cycle number . Differences
between genotypes were assessed using an unpaired, two-tailed
OB tissue of wild-type animals and Dab1 mutant mice (3 month
of age) was homogenized in ice-cold lysis buffer (20 mM Tris-HCl,
0.15 M NaCl, 2 mM EDTA, pH 7.5, protease inhibitor mixture;
Roche Diagnostics). Lysates were cleared two times by centrifu-
gation (10,000 g, 5 min, 4uC). The soluble cytosolic fraction was
removed after ultracentrifugation (200,000 g, 30 min, 4uC) and
pellets resolved in lysis buffer containing additional 0.1% sodium
dodecyl sulfate and 1% Trition-X-100. Soluble membrane
fractions were obtained by a second ultracentrifugation step
(100,000 g, 30 min, 4uC) and analyzed by standard SDS-PAGE
and Western immunoblotting using chemoluminescence detection
techniques (Super Signal West Pico, Perbio Science). Specificity of
Vldlr antibodies was confirmed by loading cell lysates of NIH 3T3
fibroblasts transfected to express Vldlr-Dab1-GFP-fusion protein
For immunoblotting the following primary antibodies were
used: anti-Vldlr (1:1,000, a kind gift from A. Goffinet), anti-Crmp-
1 (1:1,000, AB9216, Millipore) and anti ß-actin (1:5,000, A2066,
Sigma-Aldrich). Secondary antibodies: horseradish peroxidase
(HRP) linked anti-mouse (1:10,000, NA931V, Amersham Bios-
ciences) and HRP linked donkey anti-rabbit (1:10,000, NA934V,
In situ Hybridization
reverse-transcribed using reverse transcriptase (Superscript II,
Invitrogen) and used as a template for PCR reactions.
The following oligonucleotide primers containing additional
T3/T7 binding sites were used to amplify a specific 330 BP Vldlr
DNA fragment: fwd 59-GCAGATGAGTTCACTTGCTCC-39
and rev 59-CCTTGCAGTCAGGGTCTCC-39). The PCR prod-
uct was purified and sequenced before in vitro transcription. For
other templates plasmid DNA was linearized with the appropriate
enzyme and purified.
In vitro transcription.
In vitro transcription was performed
using 1 mg linearized plasmid DNA or PCR template in the
presence of digoxigenin-11-UTP (DIG labeling mix, Roche),
RNasin (Promega), 16 transcription buffer and T3, SP6 or T7
RNA polymerase (Roche Diagnostics). The following DIG-labeled
riboprobes were generated: Dab1 , ApoER2 , Crmp-1 (see
Allen Brain Atlas, http://mouse.brain-map.org, Ref. 73512561)
RNA detection by in situ hybridization.
brains were fixed in freshly prepared 4% PFA in PBS,
cryoprotected in 30% sucrose and frozen in isopentane at
260uC. In situ hybridization was performed on 50 mm free-
floating cryostat sections as described previously .
Total RNA from P0 wild-type brain was
Cultures of SVZ-explants (n=3–5 for each genotype) were
performed as described previously . P2 brains were dissected
and the OBs separated. The forebrains were sliced frontally into
400 mm sections in ice cold Hank9s balanced salt solution (HBSS,
Invitrogen) using a vibratome (Leica). From appropriate sections
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Reelin Signaling in the OB
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Figure 1. Expression of Reelin and components of its signaling pathway in the forebrain. (A) RT-PCR analysis at P0, P7, P14 and adult
age revealed Reelin, ApoER2, Vldlr and Dab1 mRNA expression in the OB. Samples were normalized to b-actin expression. (B) RT-PCR primers
sequences (C) Western immunoblot analysis of adult OB tissue and transfected control fibroblasts confirming protein in the OB. (D-F) At P0
strong Reelin expression is found in the mitral cell layer of the OB (open arrowheads, D). Adult mice express Reelin in the MCL (open
arrowhead) and in a periglomerular position (arrowhead, F). Reelin positive cells (arrows) are also seen in close vicinity to the rostral migratory
stream (RMS, E). Migrating precursors in the RMS are negative at both analyzed timepoints. (G-I) Early postnatal Vldlr expression is restricted to
the MCL (open arrowhead, G). At adult age Vldlr mRNA is found in the RMS (arrows, H and insert, arrowhead), in the MCL (open arrowheads, I)
and in the innermost part of the glomerular cell layer (GL), (arrowheads, I). Similar expression patterns were obtained by in situ hybridization for
ApoER2 (K-L) and Dab1 (N, O) on sagittal sections of adult wt forebrains. Of note, both are expressed in the RMS at P0 (J, M) and migrating
precursors. Scale bars: D, G, H, J, K, M, N: 200 mm; E, F, I, L, O: 100 mm.
Figure 2. The Reelin detachment signal in vitro is mediated by ApoER2. (A) Experimental setup: co-culture of OB and explants from the
subventricular zone (SVZ). After 48 hours all analyzed explants showed radial chain migration when cultured without OB. No differences were
observed comparing the analyzed mutants with control animals (wild-type (wt, B), Vldlr2/2(D), ApoER22/2(F) and Dab12/2(H) ). Co-culture of SVZ
explants and OB induced detachment of neuroblasts from the explant in wt mice (C) and Vldlr2/2(E) and Dab12/2mutants (I). SVZ-cultures from
ApoER22/2mice (G) remained unaffected (insert: higher magnification of another ApoER22/2explant). (J, L, N) Quantification of the relative migration
distance (K, M, O) Quantification of the relative number of single cells per field. Statistical analysis by using Wilcoxon-Mann-Whitney test with
*indicating significant difference. Data expressed as mean 6 SEM. Scale bars: A: 200 mm; all others: 50 mm.
Reelin Signaling in the OB
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Figure 3. Functional integrity of the rostral migratory stream (RMS) and detachment process in vivo. Immunohistochemistry for PSA-
NCAM (red), GFAP (green) and DAPI on sagittal sections through the forebrain of adult wild-type mice, Vldlr2/2, ApoER22/2, Dab12/2, reeler and
ApoER22/2:Vldlr2/2mutant animals show the presence of neuroblasts proximal to the SVZ (arrows) (column A) and approaching the OB (open
arrowheads) (column B) in all animals analyzed. Higher magnification on frontal sections and PSA-NCAM staining revealed a proper detachment of
neuroblasts with the typical switch from chain migration to radial ascension into layers in wild-type mice, Vldlr2/2and ApoER22/2mutants (column
C). Single cells extend their leading process towards outer layers (open arrowheads, dashed arrow indicates migration direction from the RMS
towards distal/upper layers). In contrast, reeler mutants as well as double receptor knockout mice exhibit a severe impairment of the detachment
process (column C). Dab12/2animals appear to have only a minor phenotype (arrowheads). In (D) the number of BrdU labeled nuclei 16 days after
BrdU injection in different areas of the OB (core (proximal to the RMS), GCL (granule cell layer), MCL (mitral cell layer) and EPL (external plexiform
layer) area and GL (glomerular cell layer) are shown. Numbers are given as BrdU+/total cells [%]. Student9s test compared number of BrdU+in mutants
to corresponding region of wild-type animals. *p,0.05; **p,0.01. V=ventricle; Scale bars: column A 100 mm, column B 200 mm and column C
Reelin Signaling in the OB
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Figure 4. Labeling of early generated interneurons. Immunohistochemistry for Calbindin on frontal sections through the OB of adult wild-type
mice showed strong expression in the glomerular cell layer (GL, column A, wt). A similar staining pattern was observed in Vldlr2/2mice (column A,
Vldlr2/2) although few positive cells hosted in the external plexiform layer. ApoER22/2, Dab12/2, reeler and ApoER22/2:Vldlr2/2mutants show clearly
two separate cell populations using Calbindin as marker (column A). A superficial one in the typical periglomerular position and a deep one in the EPL
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the SVZ was dissected out and cut into smaller pieces (100–
300 mm in diameter). After mixing the SVZ-explants with
Matrigel (Becton Dickinson) and incubation at 37uC for 10 min
the polymerized gel was covered with 500 ml nutrition medium
(Neurobasal A, 2% B27 supplement, 50 U/ml penicillin, 50 mg/
ml streptomycin, 0.5 mM L-glutamine, Invitrogen). For culturing
SVZ-explants uncoated glass cover slips (12 mm) were placed into
non-treated 4 well dishes (179820, Thermo Fisher Scientific). For
co-culturing experiments pieces of OB (approximately 300 mm
diameter) were placed next to the SVZ-explants before polymer-
ization of the matrigel. The cultures were maintained in
a humidified atmosphere with 5% CO2at 37uC for 2 days. After
48 h explants were monitored and the number of individual
neurons per field and the length of migratory chains were
measured using AnalySIS image analysis software (SIS). Statistical
analysis was performed by using the non-parametric Wilcoxon-
Mann-Whitney Test. Significance was assigned for all tests at
and sliced sagittally on a vibratome (50 mm). Sections were pre-
incubated for 60 minutes in blocking solution (10% fetal calf serum
(FCS) in 16 PBS) at room temperature. Subsequently, sections
were incubated with the primary antibodies in 3% FCS containing
0.1% Triton-X 100 in 16 PBS overnight at 4uC. For staining
against PSA-NCAM Triton-X 100 was avoided. After washing
(3610 min, PBS) at room temperature, sections were incubated
with secondary fluorochrome-conjugated antibodies (Alexa-Fluor
series, Invitrogen) for 2 hours at room temperature. After rinsing
in PBS (three times, 30 min) sections were mounted in fluorescent
mounting medium (DAKO) supplemented with 1 mg/ml 49, 6-
The following antibodies were used: anti-Reelin
(1:250, Millipore, MAB5364); anti-Calbindin, anti-Calretinin and
anti-Parvalbumin (each 1:1,000, Swant, CB-38a, 7699/4, PV25),
anti-BLBP (1:500, Millipore, AB9558), anti-Polysialic acid (1:200,
Millipore, MAB5324), anti-GFAP (1:1,000, DAKO, Z0334),
rabbit anti-Tyrosin Hydroxylase (1:1,000, Millipore, AB152),
and anti-Tbr1 (1:5,000, Millipore, AB9616). Secondary antibo-
dies: Alexa-Fluor series (Invitrogen: A-11004, A-11008, A-11011,
A-21043, each antibody 1:1000).
Quantification and statistical analysis.
Calbindin-positive interneurons was obtained from 10 section per
OB (n=324 for each genotype), respectively. Cell number was
estimated in the external plexiform layer. The area of interest was
measured by using AnalySIS- software (SIS). Statistical analysis
was performed by using the non-parametric Wilcoxon-Mann-
Whitney Test. Significance was assigned for all tests at p,0.05.
Brains were fixed in 4% PFA in PBS
The number of
BrdU Injection and Staining
Six to eight weeks old mice (n=325 animals of each genotype)
were injected intraperitoneally with a sterile solution of BrdU
(50 mg/kg, Roche Diagnostics) in PBS. Brains were fixed in
freshly prepared 4% paraformaldehyde (PFA) in PBS in paraffin
embedded and cut into 7 mm serial sagittal sections using
a microtome (Leica). For BrdU detection sections were de-
paraffinized, antigen-recovery protocols using acidic and alkaline
treatment applied and sections stained as previously described
. A monoclonal mouse anti-BrdU antibody (1:1,000, Roche
Diagnostics) was used to detect BrdU incorporation. Sections were
exposed to secondary antibodies (Alexa-Fluor series, Invitrogen)
and finally embedded in fluorescent mounting medium (DAKO).
The number of BrdU-positive cells was counted on eight sagittal
sections through the OB and RMS per genotype. The OB was
divided into three areas (core, medium incl. GCL, MCL and EPL
and finally the glomerular cell layer). Of each area on each section
200–400 cells (DAPI positive nuclei) were counted and the number
of BrdU positive cells determined and expressed as number of
BrdU positive cells per 100 total cells.
Expression of Reelin, ApoER2, Vldlr and Dab1 in the OB
We analyzed mRNA expression of Reelin and components of its
signaling pathway in the OB by semi-quantitative RT-PCR at
different developmental time points (P0, P7, P14 and adult). Reelin
mRNA was present in OB samples at all developmental stages
analyzed (Fig. 1A). Similar results were found for Vldlr, ApoER2
and Dab1 (Fig. 1A). Western immunoblotting analysis confirmed
the presence of Vldlr protein in the adult OB of wild-type mice
(Fig. 1C).To study the corresponding expression patterns, we
performed immunohistochemistry for Reelin and in situ hybrid-
ization experiments for ApoER2, Vldlr and Dab1 on sagittal brain
sections of early postnatal (P0) and adult wild-type mice. At P0
Reelin was strongly expressed in the MCL likely by mitral cells
(Fig. 1D and F) whereas Reelin positive interneurons were
detected in the innermost part of the GL of adult animals
(Fig. 1F). At both time-points Reelin was not present in the RMS
(Fig. 1D, E). However, some Reelin-positive cells were observed in
close proximity to the RMS (Fig. 1E).
At early postnatal stages weak expression of Vldlr was observed
in the MCL while the RMS remained negative (Fig. 1G). Of note,
in the adult animal migrating neuroblasts in the RMS were Vldlr
positive (Fig. 1H). In the adult OB Vldlr (Fig. 1I), ApoER2 (Fig. 1L)
and Dab1 (Fig. 1O) expression were present within the MCL and
in a periglomerular position. ApoER2 and Dab1 were expressed in
the mitral cell layer at P0 and by migrating neuroblasts in the
RMS at both developmental time points (Fig. 1J, K, M, and N).
The Reelin Detachment Signal is Mediated via ApoER2
Reelin induces dispersal of chain migrating neuroblasts from
SVZ-explants . To investigate in more detail if components of
the Reelin signaling cascade participate in the detachment process
we cultured SVZ-explants of control animals, Vldlr2/2, ApoER22/
2and Dab12/2animals in a three dimensional extracellular
matrix (Matrigel). This set up allows the analysis of chain
migrating neuroblasts in vitro in the presence or absence of OB
tissue, known to secrete Reelin. After 48 hours of culturing, all
explants showed symmetrical radial chain migration of neuroblasts
when cultured without OB tissue (Fig. 2B, D, F, H). In contrast,
SVZ explants of wild-type (Fig. 2C), Vldlr2/2(Fig. 2E) and Dab12/
2mutants (Fig. 2I) cultured in the presence of OB tissue rarely
showed migration of neuroblasts in chains. Precursor cells that
(arrowheads, column A). (Column B) Immunohistochemistry for Parvalbumin on frontal sections through the OB of adult mice labeled interneurons in
the external plexiform layer (EPL). No differences were observed for the analyzed mutants compared to wild-type mice. Staining for Tbr1 (column C)
and Reelin (column D) as markers of the MCL showed no alterations in the analyzed animals except the loss of Reelin immunoreactivity in reeler
mutants. Quantification of the relative number of Calbindin positive (E) cells per area in the EPL after normalization to the wt situation (n=325
animals per genotype). Wilcoxon-Mann-Whitney test; Data expressed as mean 6 SEM. Scale bar 100 mm. MCL, mitral cell layer; GCL, granule cell layer.
Reelin Signaling in the OB
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migrated out of the explants dispersed and lost contact with the
explant or with other cells. Importantly, co-culturing of SVZ tissue
from ApoER22/2mutants (Fig. 2G and insert) with OB tissue did
not affect the radial chain migration. For quantification of the
chain length of the migrating neuroblasts and the effect on
dispersion induced by the presence of Reelin secreting OB tissue
see (Fig. 2J–O).
These results indicate that the detachment signal in explant
cultures requires ApoER2 and, unexpectedly, this process appears
to be independent of Vldlr and Dab1.
All Mutant Animals Exhibit an RMS and Show BrdU
Incorporation into the OB
We examined the RMS in the various mutant animals as
a prerequisite for correct interneuron turnover in the OB. We
performed first immunohistochemistry for PSA-NCAM a marker
of neuroblasts on sagittal sections of adult brains of wild type and
mutant animals (Fig. 3). We found that all animals analyzed
exhibited PSA-NCAM positive neuroblasts proximal to the SVZ
(Fig. 3 column A) as well as distal when the RMS enters the OB
(Fig. 3 column B). Next, to analyze whether neuroblasts actually
enter the OB, we performed BrdU pulse labeling experiments to
label newborn cells and analyzed adult mice 16 days after a single
BrdU injections. We found BrdU immunoreactivity located within
the OB in all analyzed mutant and wild-type animals revealing
a functional RMS. BrdU positive cells were found in the core
region at intermediate positions (GCL, MCL and EPL area) as
well as in the GL of each analyzed OB (pictures not shown).
Quantifications of BrdU positive cells and statistical analysis are
shown in Fig. 3D. Of note, the reeler mutant as well as the
ApoER22/2and ApoER22/2:Vldlr2/2double receptor knockout
mutant mice showed a marked reduction in BrdU positive cells in
all OB regions. In contrast, Vldlr2/2mutant mice exhibited BrdU
incorporation within the OB similar to wild-type animals.
Interestingly, Dab12/2animals showed no reduction in BrdU
staining in the core region of the OB and a significant reduction in
outer layers. These data substantiate the findings of our culturing
experiments that ApoER2 is the main receptor for Reelin
mediated detachment of neuroblasts from chain migration into
radial migration independent of Vldlr.
Detachment of Neuroblasts in the Adult OB of ApoER2
Mutants is not Altered
Our in vitro data point towards ApoER2 as being the main
receptor in the detachment process of neuroblasts within the OB.
Therefore, we studied next the detachment process in vivo by
performing immunohistochemistry for PSA-NCAM on frontal
Figure 5. Labeling of Calretinin-positive interneurons in the
granule cell layer (GCL) at different developmental timepoints.
Immunostaining for Calretinin on frontal sections through the OB. Wild-
type (A-C) and Vldlr2/2mice (D-F) at P7 and P14 and adult age reveal
the typical formation of Calretinin positive interneurons in the GCL, with
a radial orientation of neurites towards upper layers (arrowheads). In
reeler (M-O) and ApoER22/2:Vldlr2/2double KO animals (P-R) a severe
disruption of the GCL is observed. Calretinin-positive interneurons
accumulate in clusters and the radial orientation of dendrites is lost
(arrowheads). In ApoER22/2mutants a reeler-like phenotype is found
one week after birth (G). However, at P14 a compact layer begins to
form with few cells extending their neurites towards the surface (H),
while adult ApoER22/2mice (I) exhibit minor alterations compared to
wild-type mice. Dab12/2animals (J-L) exhibit a weak reeler like
phenotype at P7 and P14 with less strict orientation of neuritis towards
upper layers (arrowheads, J and K). At adult stage Dab12/2mice appear
similar as wild-type animals (L). Scale bar: 50 mm. and Dab12/2mice (J-
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sections of adult OBs. We observed the characteristic switch from
tangential to radial migration associated with the presence of
a characteristic leading processes oriented towards OB layers in
wild-type animals, Vldlr2/2and ApoER22/2mutants (Fig. 3
column C). In contrast, reeler mutants show a severe impairment in
the switch from chain to radial migration, which occurred to be
phenocopied in ApoER22/2:Vldlr2/2double receptor knockout
mutants (Fig. 3 column C). In these mutants leading processes
were not oriented, hardly visible and the cells appeared not
organized. Dab1 deficient mutants showed an intermediate
phenotype. Here, the leading processes were often visible but
not as strictly oriented towards outer layers and the cells appeared
less organized than in wild type mice. However, the Dab12/2
phenotype was not as strong as in reeler and double-knockout
Formation of OB Layers in Mutants of the Reelin
To elucidate the role of Reelin and components of its signaling
cascade in the formation of OB layers we used interneuron and
mitral cell markers to examine their position within the OB of wild
type, Vldlr2/2, ApoER22/2, Dab12/2, reeler and ApoER22/
2:Vldlr2/2double mutant animals.
The calcium binding protein Calbindin is
expressed by distinct interneuron populations of the glomerular
cell layer. Thus, in wild-type mice Calbindin-positive somata are
found in a periglomerular position. Cells extended their dendritic
processes to form the characteristic spherical structure of
a glomerulus (arrow, Fig. 4 column A). Only a few cells were
distributed throughout other layers. A similar normal distribution
of Calbindin-positive interneurons was observed in Vldlr2/2
mutants (Fig. 4 column A) although a few but statistical significant
number of Calbindin-positive interneurons were found in the EPL
(see also Fig. 4E). In contrast, reeler, ApoER22/2, Dab12/2and
ApoER22/2:Vldlr2/2mutant animals exhibited a more dramatic
phenotype with the appearance of many mispositioned Calbindin
positive cells in the EPL. In ApoER22/2mutants the majority of
the Calbindin-positive interneurons were located correctly in the
GL. However, a large subset of mispositioned interneurons was
found in the inner most part of the EPL adjacent to mitral cells
(arrowheads, Fig. 4 column A, ApoER22/2). Dab1-deficient
animals, reeler mutants and double receptor knockout mice showed
similar alterations in Calbindin-positive interneuron distribution
compared to ApoER22/2mice (arrowheads, Fig. 4 column A).
Quantification of the mispositioned Calbindin-positive interneur-
ons revealed that ApoER22/2single and ApoER22/2:Vldlr2/2
double knock out, Dab12/2and reeler mutants exhibited the most
pronounced phenotype regarding misplaced cells within the MCL
compared to wild type and Vldlr2/2mutants (Fig. 4E). We also
analyzed Tyrosine Hydroxylase (TH) positive cells representing
a second distinct interneuron population located within the GL.
Similar to Calbindin-positive interneurons the TH-positive pop-
ulation exhibits localization to the GL in wild type animals and
mispositioning of cells in the EPL in ApoER22/2single and
ApoER22/2:Vldlr2/2double knock-out animals (Fig. S3A). The
quantification of mispositioned TH positive interneurons revealed
no aberrant changes in the EPL of Vldlr2/2mutants compared to
External plexiform layer (Parvalbumin).
Parvalbumin-positive cells of the EPL (mostly van Gehuchten cells)
revealed no alterations in all mutant animals analyzed. All mutants
exhibited the appearance of Parvalbumin positive interneurons in
the EPL as seen in wt animals (arrowheads Fig. 4 column B).
The analysis of
Mitral cell layer (Reelin and Tbr-1).
principal projection neurons are the major source of Reelin
expression in the OB. Accordingly, Reelin served as marker for
this cell population. Despite the lack of Reelin expression in the
mitral cell layer in reeler mutants no alterations were found when
analyzing adult receptor single and double knockout mice as well
as Dab1 deficient mutant animals when compared to wild-type
animals (Fig. 4 column D). Tbr-1 was used as additional mitral cell
marker revealing a comparable distribution in the MCL in all
mutant animals as seen in wild type (Fig. 4 column C).
Granule cell layer: Calretinin.
tein Calretinin is expressed by subsets of interneurons in the GCL
and GL of the OB . Here it served as marker to examine the
morphology of the GCL at different developmental time points
(P7, P14, adult). In wild-type animals the Calretinin-positive
interneurons of the granule cell layer show a compact organization
with strictly oriented neurites projecting towards the superficial
layers at all time-points analyzed (Fig. 5A–C with arrowheads
labeling neurites). In contrast, Calretinin immunostaining of reeler
mutants revealed a severe disruption of the GCL (Fig. 5M–O).
Many Calretinin-positive interneurons appeared less confined to
a particular region of the GCL and were distributed over the
entire GCL, formed clusters and were often lacking extending
dendrites and radial orientation of dendrites towards the EPL. The
severity of this phenotype increased with age. A similar a reeler-like
phenotype was observed in ApoER22/2:Vldlr2/2double-knockout
mice with clusters and mispositioned interneurons within the GCL
(Fig. 5P–R). Surprisingly, immunohistochemical analysis for
Calretinin in Dab12/2animals (Fig. 5J–L) revealed only a weak
phenotype at P7 and P14. No clusters were observed and the
neurons showed oriented processes but with less strict orientation
as seen in wild type animals. However, at adult stage the
phenotype of Dab12/2animals appeared similar than the one
observed in wild type.
These observations suggest that one or maybe both of the
Reelin receptors participate in Reelin-mediated layering. To
clarify this question we analyzed Vldlr and ApoER2 single KO
animals for Calretinin expressing neurons. Vldlr2/2mutant mice
appeared to be indistinguishable from wild type animals at all ages
(Fig. 5D–F). In contrast, ApoER22/2mutants showed one week
after birth a similar aberrant morphology of Calretinin-positive
neurons as in reeler and ApoER22/2:Vldlr2/2animals (Fig. 5G, M
and P). However, at P14 the formation of a more compact granule
cell layer starts with some cells extending their neurites towards
superficial layers (Fig. 5H). At adult stage also ApoER22/2mutants
exhibit a minor phenotype reminiscent of reeler mice with some
Calretinin positive interneurons that have lost orientation whereas
the majority upholds a strict radial organization (Fig. 5I).
Investigating the Calretinin-positive cell population hosting in
the GL no abnormalities were observed in the different mutants at
adult stage (data not shown). These findings point towards
ApoER2 as being the main receptor for mediating Reelin
functions in the OB and particularly layer formation.
Mitral cells as
The calcium binding pro-
The Radial Glial Scaffold is not Altered
Our findings suggest an influence of the Reelin signaling
cascade on the migration of interneurons within the OB. We
addressed next, whether the glial scaffold was altered in the
different mutant animals, which could explain the interneuron
migration and neurite orientation phenotypes. However, perform-
ing BLBP (brain lipid binding protein) immunohistchemistry at P0
did not reveal any alterations in the organization of the OB glia
scaffold in ApoER22/2, Dab12/2, reeler and ApoER22/2:Vldlr2/2
animals (Fig. S1A–E).
Reelin Signaling in the OB
PLOS ONE | www.plosone.org9November 2012 | Volume 7 | Issue 11 | e50646
Vldlr mRNA Expression Increases with Age but is not
Altered in ApoER2 Mutants
The morphological changes in the GCL (Fig. 5) suggest for
a compensatory mechanism in ApoER22/2mutants since the
observed double receptor knockout phenotype is markedly
attenuated after P7. In view of the canonical Reelin signaling
pathway Vldlr is a possible candidate molecule to rescue the
ApoER22/2phenotype. Therefore, we examined Vldlr mRNA
changes in ApoER22/2mutant animals by quantitative RT-PCR.
We did not observe any significant changes in the expression of
Vldlr when comparing wild-type animals and ApoER22/2mutant
mice at P7 and P14, respectively (data not shown). However,
comparison of Vldlr expression levels between P7 and P14 showed
a small but significant 1.29-fold increase of Vldlr mRNA expression
in ApoER22/2animals from P7 to P14 and a 1.4-fold increase in
wild type demonstrating an age dependent increase of Vldlr mRNA
(data not shown).
Collapsin Response Mediator Protein 1 Could Act
Downstream of ApoER2 in the OB
We observed that Dab12/2neuroblasts were clearly distinguish-
able from reeler and ApoER22/2:Vldlr2/2neuroblasts regarding
detachment and migration in vitro (Fig. 3). Furthermore, adult
Dab12/2animals exhibited a normal granule cell layer. in contrast
to ApoER22/2:Vldlr2/2and reeler mice (Fig. 5) Therefore, we asked
whether other adapter molecules could mediate Reelin signaling
via ApoER2 and Vldlr. Remarkably, it has been shown that
Collapsin Response Mediator Protein 1 (Crmp1) mediates Reelin
signaling in cortical neuronal migration . In situ hybridization
for Crmp1 on sagittal sections of adult wild-type animals (Fig. S2A,
B, see also Allen Brain Atlas, http://mouse.brain-map.org, Ref.
73512561) revealed expression of Crmp1 in the rostral migratory
stream in the MCL and in a periglomerular position. In mice
deficient for Dab1, a similar expression of Crmp1 was found (Fig.
S2C). Western immunoblotting showed increased expression levels
for Crmp1 in membrane fractions of OB tissue of Dab1 mutants
compared to wild-type animals (Fig. S2D) suggesting Crmp1 as
being a candidate molecule for mediating ApoER2 and/or Vldlr
signaling in the OB.
Studies on corticogenesis have provided evidence for different
modes of neuronal migration [49,50]. Besides somal translocation
cortical neurons can use a glia-guided mode of migration to
govern the increasing distance from the germinal ventricular zone
to their definitive positions in the cortical plate . This way, the
layers of the cortex are formed in an inside-out manner [1,52].
Since aberrant migration of glia-guided neurons has been reported
in ApoER2 mutant mice a key role for ApoER2 in this process has
been proposed .
Similar to cortical lamination layer formation in the OB takes
place chronologically. First mitral cells as principal projection
neurons are generated and provide a local Reelin source .
Subsequently, interneurons follow in a sequential order . The
calbindin positive subpopulations represent early generated
interneurons. The majority of these interneurons originate from
late embryogenesis and there is a marked decline in their
generation immediately post-partum. In contrast, only few of the
Calretinin-positive interneurons are produced embryonically,
while the majority is produced after birth. Turnover of late-
generated interneurons persists throughout adulthood [2,6].
In analogy to cortical neuronal migration, glia-guided migration
has been postulated for OB layer formation . The data of our
study are consistent with this idea. The aforementioned rapid
decrease in glia is coincident with the timeframe of generation of
Calbindin positive interneurons . In the ApoER2 mutants but
not the Vldlr mutant mice migration of the early generated
Calbindin-positive subpopulations is disturbed similar to reeler and
dab2/2mutants. Furthermore, we found that also early born TH-
positive interneurons show migration defects in ApoER2 and
ApoER2:Vldlr double receptor knock-out animals but not in Vldlr
mutants. In accordance, two other observations are substantiating
the idea that Reelin-ApoER2 signaling regulates early postnatal
interneuron migration. Firstly, double-receptor-knockout mice
exhibit the same misplacement of early born interneurons.
Secondly, interneurons expressing Calretinin exhibit a strong
phenotype in ApoER22/2animals similar to reeler mice at early
postnatal stages. However, this phenotype declines with de-
velopment and adult animals show only minor alterations in the
Calretinin population compared to wild type animals. These
defects are not seen in Vldlr2/2mutants. Thus, our data support
the model that ApoER2 signaling is involved in early Vldlr-
independent migration, most likely glia guided . 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 similar to the situation seen in the developing cortex
in a yet unknown way .
A previous report claimed the absence of VLDLR protein in the
mouse OB by immunohistochemical analysis . In contrast, we
identified expression of Vldlr protein by Western immunoblotting
in the OB of adult mice and furthermore by RT-PCR and in situ
hybridization studies in postnatal and adult animals. Based on
these findings we concluded the presence of Vldlr in the postnatal
and adult mouse OB. Therefore, compensatory mechanisms
mediated by Vldlr in adult ApoER22/2animals could be
responsible for the decline and adjustment of the Calretinin
phenotype. This idea is underlined by the fact that in adult
ApoER2:Vldlr double knock out animals no compensation but
a reeler-like phenotype can be observed within the Calretinin
population of the GCL. Interestingly, we did not find defects in the
migration of Parvalbumin positive interneurons in any of our
mutant animals. Since the majority of this population shows
migration after Calbindin- but before Calretinin-positive inter-
neurons we assume that this population migrates independent of
the Reelin signaling cascade. Similarly, Tbr1- and Reelin-positive
mitral cells. These principle neurons are already present at
prenatal stages (E13) and likely migrate in a Reelin-independent
Late generated Calretinin-positive interneurons are continuous-
ly replaced throughout adulthood. Detachment of chain migrating
neuroblasts from the rostral migratory stream is accompanied by
the induction of a leading process, which directs radial individual
migration of Calretinin positive interneurons towards their final
destination . Here, Reelin has been shown to function as
detachment factor  either by acting on the interaction between
radial processes and migrating neurons or on the interaction
between apposing cells in chain migration . However, we show
that in vitro the Reelin detachment signal for neuroblasts is
mediated by ApoER2. Absence of Vldlr and surprisingly also the
intracellular adapter protein Dab1 did not influence the de-
tachment process. To analyze whether these findings are also seen
in vivo we analyzed first the presence and the functional integrity
of the RMS in all our mutants. We found that all adult mutant
strains have an RMS and showed BrdU-positive cells within the
OB 16 day after peritoneal injection of BrdU. Hence, neuroblasts
derived from the SVZ arrive and integrate in the OB of mutant
mice arguing for the presence of a functional RMS. Although
Reelin Signaling in the OB
PLOS ONE | www.plosone.org 10November 2012 | Volume 7 | Issue 11 | e50646
ApoER2, Vldlr and Dab1 are expressed within the RMS,
canonical Reelin signaling seems not to be pivotal for RMS
function . However, our immunohistochemical results re-
vealed a disruption of the GCL morphology in ApoER22/2, reeler
and double receptor knockout mice one week after birth. Since
Vldlr2/2animals do not show this phenotype at early postnatal
stages these data support our in vitro results that ApoER2 regulates
migration of Calretinin-positive neurons. Furthermore, in adult
reeler and double receptor knockout mice, PSA-NCAM positive
neuroblasts do not possess a leading process, a prerequisite for
correct migration. Moreover, most cells lose their radial orienta-
tion and accumulate in clusters. However, with advancing age the
migration defect of Calretinin-positive interneurons in ApoER2
mutants attenuates and we observed correct detachment of
precursor cells in adult ApoER22/2and Vldlr2/2mice including
leading process induction. Thus, we propose redundant function
for Vldlr and ApoER2 in the late postnatal and adult OB. The
following aspects point towards this. Firstly, the expression pattern
of Vldlr changes. While the RMS of early postnatal animals is Vldlr
negative, migrating neuroblasts in the adult OB express Vldlr
mRNA. Secondly, the Vldlr expression level increases within the
critical time between P7 and P14. Thirdly, the disruption of GCL
morphology persists in double receptor knockout mice, which
resembles the reeler phenotype.
Dab12/2mutant mice do not exhibit a detachment phenotype
in our in vitro model. Furthermore, in vivo, the detachment process
appears to be only slightly impaired similar to the formation of the
GCL. Although BrdU incorporation in outer layers of the OB was
affected in Dab12/2animals, no effect was observed in the core
region in the vicinity of the RMS. However, although the loss of
Dab1 could affect neuronal migration in a non-cell autonomous
way since Dab1 is also expressed by glia , our results suggest
also for a compensatory mechanism for Dab1 downstream of the
lipoprotein receptors. Therefore, we analyzed the expression of
candidate molecules and found Crmp1 as being very similar
expressed in the adult forebrain as Dab1. Similar to Dab1, Crmp1
has been described as an intracellular signaling mediator of Reelin
during corticogenesis . Based on its expression, Crmp1 could
also play a role in mediating Reelin signaling in the OB and
therefore being a candidate molecule responsible for the mild
phenotypes seen in Dab1 mutant animals in the OB. However,
future studies will be required to clarify this hypothesis.
Canonical Reelin and lipoprotein receptor signaling appear to
be highly redundant and compensatory mechanisms seem to
account for partially contradictionary results obtained in studies
using knock-out animal models (see e.g. study by Andrade et al.
 and the presented data). Therefore, it might be important for
future studies to emphasize genetic background variations in
individual mouse strains when comparing different mutant
animals since background variations are known to influence
redundant signaling pathways.
through the OB of wild-type (A), ApoER22/2(B), reeler (C),
ApoER22/2:Vldlr2/2(D) and Dab12/2(E) mice at P0 stained for
BLBP. No alterations were observed. GL, glomerular cell layer;
EPL, external plexiform layer; MCL, mitral cell layer; GCL,
granule cell layer. Scale bar: 50 mm.
Radial glial scaffold in the OB. Frontal sections
hybridization on sagittal brain sections revealed expression of crmp1
mRNA in the rostral migratory stream, the mitral cell layer and in
a periglomerular position in wild-type mice (A, B) and Dab12/2
mutants (C). (D) Western immunoblotting analysis of the
membrane fraction of OB tissue at P21 showed an up-regulation
of Crmp1 expression level in of Dab2/2mice compared to wild-
type animals. Blots were obtained with similar amounts of proteins
as indicated by the immunoblot for ß-actin. Molecular weights are
shown on the right of each panel. Scale bar: 100 50 mm.
Crmp1 expression in the forebrain. In situ
neurons. Immunohistochemistry for TH on frontal sections
through the OB of adult wild-type mice showed strong expression
in the glomerular cell layer (A, GL, wt). A similar staining was
observed in Vldlr2/2mice (A, Vldlr2/2) although some cells were
tions (A). A typical superficial periglomerular labeling and a deep
layer staining in the EPL (A, arrowheads). (B) Quantification of the
relative number of TH positive cells per area in the EPL after
normalization to the wt situation (n=3–5 animals per genotype)
shows a significant quantitative mispositioning of TH-positive
but not in Vldlr2/2animals. Wilcoxon-Mann-Whitney test; Data
expressed asmean 6 SEM.Scale bar100 mm.
Labeling of early generated TH-positive inter-
The authors thank Dr. Michael Frotscher for discussions, support and the
possibility to carry out this work. Furthermore we thank Jutta Peschke and
Ruhtraut Ziegler for excellent technical assistance and Dr. Verdon Taylor
for critical reading of the manuscript.
Conceived and designed the experiments: SH DJ. Performed the
experiments: SH IH BZ BB DJ. Analyzed the data: SH BZ DJ.
Contributed reagents/materials/analysis tools: SH IH BZ BB DJ. Wrote
the paper: SH DJ.
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Reelin Signaling in the OB
PLOS ONE | www.plosone.org12 November 2012 | Volume 7 | Issue 11 | e50646