© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 4318-4328 doi:10.1242/dev.123943
RESEARCH ARTICLE
Tag1 deficiency results in olfactory dysfunction through impaired
migration of mitral cells
George G. Bastakis1, *, Maria Savvaki1,*,‡, Antonis Stamatakis2, Marina Vidaki1 and Domna Karagogeos1,‡
The olfactory system provides mammals with the abilities to
investigate, communicate and interact with their environment.
These functions are achieved through a finely organized circuit
starting from the nasal cavity, passing through the olfactory bulb and
ending in various cortical areas. We show that the absence of
transient axonal glycoprotein-1 (Tag1)/contactin-2 (Cntn2) in mice
results in a significant and selective defect in the number of the
main projection neurons in the olfactory bulb, namely the mitral cells.
A subpopulation of these projection neurons is reduced in Tag1deficient mice as a result of impaired migration. We demonstrate that
the detected alterations in the number of mitral cells are well
correlated with diminished odor discrimination ability and social
long-term memory formation. Reduced neuronal activation in the
olfactory bulb and the corresponding olfactory cortex suggest that
Tag1 is crucial for the olfactory circuit formation in mice. Our results
underpin the significance of a numerical defect in the mitral cell layer
in the processing and integration of odorant information and
subsequently in animal behavior.
KEY WORDS: Mitral cells, Migration, Olfaction
INTRODUCTION
The olfactory system can discriminate and process a variety of odors
through its fine and sophisticated structure (Fig. 1A). Olfactory
sensory neurons (OSNs) from the olfactory epithelium (OE) project
to the main olfactory bulb (MOB), where they synapse with
dendrites of mitral and tufted cells (MCs, TCs), the olfactory
projection neurons, and form the glomeruli structures (Whitman
and Greer, 2009). Each OSN expresses a unique type of receptor and
projects towards topographically restricted glomeruli (Buck and
Axel, 1991; Mombaerts et al., 1996; Mombaerts, 2001; Mori et al.,
2006; Sakano, 2010). MCs and TCs from the MOB project their
axons towards the lateral olfactory track (LOT), and transmit the
olfactory signal to divergent areas of the olfactory cortex ( piriform
and entorhinal cortices, olfactory tubercle, etc.) (Mouret et al., 2009;
Leinwand and Chalasani, 2011). The accessory olfactory system
(AOS), which is responsible for pheromone perception, is similarly
organized, with the primary sensory neurons of the vomeronasal
organ projecting to the accessory OB (AOB, dorsal OB region).
Subsequently, the MCs and TCs of the AOB send their axons
through the LOT to cortical regions (Baum and Kelliher, 2009;
1
Department of Basic Science, Faculty of Medicine, University of Crete and Institute
of Molecular Biology and Biotechnology-FoRTH, Vassilika Vouton, Heraklion,
2
Crete 71110, Greece. Laboratory of Biology, Faculty of Nursing, School of Health
Sciences, University of Athens, Papadiamantopoulou 123, Athens GR11527,
Greece.
*These authors contributed equally to this work
‡
Authors for correspondence ([email protected]; [email protected])
Received 4 March 2015; Accepted 22 October 2015
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de Castro, 2009). Olfactory system dysfunction has been described
in aged individuals as well as in patients with neurodegenerative
disorders and dementias. Recent studies propose it as an early
biomarker for the diagnosis and progression of these diseases
(Attems et al., 2014).
MCs are generated in the ventricular zone of the rostral
telencephalon during embryonic days (E)9.5-13.5 and migrate
radially towards the intermediate zone (IZ), where they acquire a
tangential-like morphology (Blanchart et al., 2006; Imamura et al.,
2011; Imamura and Greer, 2013). The precise mechanisms
underlying MC development are not yet understood. The
adhesion molecules neuropilin 1 and 2 (Nrp1/2), L1 (also known
as L1cam), Ocam (also known as Ncam2) and transient axonal
glycoprotein-1 (Tag1), also known as contactin-2 (Cntn2), are
expressed on OB projection neurons during development but their
involvement in the integration of projection neurons into the OB
circuitry is not yet elucidated (Wolfer et al., 1994; Yoshihara et al.,
1995; Treloar et al., 2003; Inaki et al., 2004). Here we analyze the
role of Tag1 in OB development, organization and function. Tag1,
an immunoglobulin superfamily (IgSF) member, is implicated in
key processes such as axonal fasciculation, pathfinding, neurite
extension, neuronal migration and in the maintenance of the
integrity of myelinated fibers (Furley et al., 1990; Denaxa et al.,
2001; Karagogeos, 2003; Traka et al., 2003; Baeriswyl and
Stoeckli, 2008; Wolman et al., 2008).
Tag1 is expressed by OB projection neurons and is detected on
the axons that occupy the superficial layer of the LOT during
development (Inaki et al., 2004). Our analysis revealed reduced
numbers of MCs in the MOB of Tag1-deficient mice, attributed to
altered distribution resulting from abnormal migration of a specific
neuronal subpopulation, born at E11.5. The cellular defects are
accompanied by behavioral deficits related to olfaction and reduced
neuronal activity of the main olfactory system (MOS). Our data
support a crucial role for Tag1 in the correct positioning of MCs, a
prerequisite for proper OB function. Our study further indicates that
disruption in a subpopulation of MCs can result in severe olfactory
deficits.
RESULTS
Tag1 expression in the developing olfactory system
We analyzed the spatiotemporal expression of Tag1 in the
developing OB and correlated it with specific markers. Previous
reports show expression by MCs and TCs and their axons in the
developing OB (Wolfer et al., 1994; Yoshihara et al., 1995; Inaki
et al., 2004). Tag1 is detected as early as E12.5 in the OB and the OE
(Fig. S1A-D). The expression in the OB is high at E13.5 and E15.5
and decreases towards birth (Fig. S1C,E,G). Tag1 protein and its
mRNA are strongly present in the IZ of the OB primordium at E12.5
and E13.5 which contains mainly the premature MCs (Fig. S1A,C).
With the gradual maturation of OB structure, Tag1 is specifically
expressed by migrating MCs and TCs detected in the mitral cell
DEVELOPMENT
ABSTRACT
RESEARCH ARTICLE
Development (2015) 142, 4318-4328 doi:10.1242/dev.123943
Fig. 1. Tag1 expression in the developing olfactory system. (A) Schematic representation of the olfactory system organization in rodents. Blue, main
olfactory system (MOS); red, accessory olfactory system (AOS). (B) Design of Tag1 loxP-GFP-loxP-DTA mouse expressing GFP under the Tag1 gene promoter.
(C) Analysis of Tag1 expression in the OB and OE. (Ci,v) IHC for GFP and Tag1 at E13.5 OB and OE respectively. There is co-localization of GFP and Tag1 in the
intermediate zone (IZ) of the OB (i) and in the olfactory nerve (asterisks in v), whereas GFP expression is found in the OSNs inside the OE. GFP co-localizes with
Tbr1 in E14.5 OB (ii), but not with Olig2 (iii) or GABA (iv), which are markers of oligodendrocytes and interneurons, respectively, at P0 in OBs. (Cvi-viii) Fluorescent
ISH at E16.5 OE. Tag1 is co-expressed with Gap43 and rarely with Omp (asterisk in vi). It also co-localizes with Mash1 (vii), but not with neurogenin 1 (Ngn1, viii).
Scale bars: 100 μm in Ci,v; 50 μm in Cii-iv,vi-viii. AMY, amygdaloid nuclei; AOB, accessory olfactory bulb; AON, anterior olfactory nucleus; BST, bed nucleus of
the stria terminalis; EC, entorhinal cortex; LOT, lateral olfactory tract; MOB, main olfactory bulb; nAOT, nucleus of the accessory olfactory tract; OB, olfactory bulb;
OT, olfactory tubercle; PC, piriform cortex; TT, tenia tecta; SEZ, subependymal zone.
OSNs are unaffected but MCs of the MOB are fewer in Tag1 −/−
mice
We then analyzed the phenotype of Tag1-deficient mice in the
olfactory system. As Tag1 is expressed early in OSN development,
we tested whether mature OSNs are affected. ISH for Omp revealed
no difference in the number of mature OSNs in deficient (Tag1 −/−)
vs wild-type (WT) controls (Fig. 2A-C). No gross abnormality
in the projections of OSNs to the glomeruli in adult Tag1 −/− mice
was detected by Ocam immunoreactivity (Fig. S2A,B). Previous
studies have shown that the genes encoding the adhesion molecules
Kirrel2 and Kirrel3 are transcribed in an activity-dependent manner
(Serizawa et al., 2006; Kaneko-Goto et al., 2008). We performed
immunohistochemistry (IHC) for Kirrel2 combined with olfactory
receptors mOR-EG or OR-I7 (also known as Olfr73 and Olfr2,
respectively) in order to analyze the glomerular map formation and
activity (Fig. S2C-R). We detected no changes between Tag1 −/−
mice and WT controls.
We looked closely at MCs and TCs in the OB of Tag1 −/− mice.
TC density in adult Tag1 −/− animals presents no defects by reelin
IHC (Fig. 2D-F). By contrast, we detected a gross reduction in the
number of MCs distributed in the MCL of adult Tag1−/− mice, using
the MC-specific marker Tbx21 (Fig. 2G,H). Quantification revealed
a significant dose-dependent reduction of 14.2% in heterozygous
mutants (Tag1+/−) and 28.7% in Tag1−/− mice compared with
control age-matched WT mice (Fig. 2I). In newborns (P0), a 24.8%
reduction in the number of MCs in the MCL of Tag1−/− mice
compared with WT is detected, suggesting that the defect occurs
during development (Fig. 2J-L).
The main stream of MCs, born at E11.5 are reduced in the
MCL of Tag1 −/− mice and ectopically located in the
developing AOB
The majority of MCs in the OB originate at E11.5, whereas cells
born earlier (E9.5 and E10.5) and later (E12.5 and E13.5) give rise
to fewer MCs (Hinds, 1968a,b; Imamura et al., 2011). We injected
Tag1 +/+;GFP pregnant mice intraperitoneally with BrdU at E10.5,
11.5 and 12.5 and sacrificed at P0, when the organization of the OB
is almost complete. We show that BrdU+ cells born at E11.5 and
E12.5 co-localize with GFP at the MCL, whereas cells born at E10.5
do not (Fig. 3A-C). In order to identify the subpopulation of MCs
whose localization is affected by Tag1 loss, we performed a
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DEVELOPMENT
layer (MCL) of the MOB, in the internal plexiform layer and the
periventricular part of the OB (Fig. S1E,G,H). In the OE, Tag1
mRNA expression in the vomeronasal organ reaches high levels
around E15.5, mainly at the dorsal part, and is significantly reduced
at birth [ postnatal day (P)0] (Fig. S1D,F,G). No expression is
detected in the AOB (Fig. S1H, asterisks). Tag1 is also detected in
the anterior olfactory nucleus, although there is no expression inside
the piriform cortex (PC, layers II/III), olfactory tubercle and nucleus
accumbens (Fig. S1I-L).
In the OB, Tag1 protein is localized in the cell body until E16.5,
although after E17.5 it is mainly detected on the axon (Fig. 1A,C;
Fig. S1). In order to track and study Tag1 expression in vivo, we
generated a transgenic mouse line that expresses green fluorescent
protein (GFP) under the control of the Tag1 gene promoter
(Tag1 loxP-GFP-loxP-DTA; Fig. 1B). GFP recapitulates Tag1 expression
in the OB of E13.5 mice (Fig. 1Ci). It is detected in the IZ at E14.5,
on immature MCs and TCs (co-expression with the early marker
Tbr1 in Fig. 1Cii), but not on oligodendrocytes or interneurons in
newborn OBs (co-expression with Olig2 and GABA, markers of
oligodendrocytes and interneurons, respectively; Fig. 1Ciii,iv). In
the OE, GFP is detected in OSNs and co-localizes with Tag1 only in
the olfactory nerve (Fig. 1Cv, asterisks). Triple fluorescent in-situ
hybridisation (ISH) for Tag1, Gap43 (marker of immature OSNs)
and Omp (marker of mature OSNs), showed that Tag1 is
predominantly expressed by immature neurons in the OE and is
rarely detected in mature OSNs (Fig. 1Cvi, asterisk). Subsequent
analysis for two of the main subgroups of OSNs revealed the
selective expression of Tag1 by Mash1 + (also known as Ascl1) and
not by neurogenin1 + (Ngn1, also known as Neurog1) neurons
(Fig. 1Cvii,viii).
RESEARCH ARTICLE
Development (2015) 142, 4318-4328 doi:10.1242/dev.123943
thorough analysis for BrdU (injected at E11.5 and E12.5,
respectively) on P0 OB sections from WT and Tag1−/− mice
(Fig. 3D,E, Fig. S3). A severe reduction (39.4%) in the number of
E11.5-born cells is detected at the MCL of the MOB in Tag1−/−
animals (Fig. 3F), whereas this was not the case for E12.5-born MCs
(Fig. S3). Our results suggest that MCs of the MOB, born at E11.5,
comprise the main population affected by Tag1 deficiency.
Tag1 loss could affect migration or guidance of MCs, the pool of
MC progenitors ( proliferation, specification or survival), postmitotic differentiation, or a combination of the above. We found that
the number of immature Tbr1+ projection neurons is unaffected at
E14.5 and that limited cell death is detected in both genotypes by
Caspase3 IHC at different developmental stages (E14.5-E18.5)
(Fig. S4 and data not shown).
In order to test for mislocalization of MCs, we looked for E11.5born MCs in the whole volume of the OB. Tag1 is not expressed in
the AOB, which is located on the dorsolateral part of caudal OB.
However, the number of E11.5-born (BrdU-labeled) cells found in
this area was increased in Tag1−/− mice at P0 and E17.5 (Fig. 3G,I),
although BrdU+ cells were reduced in the MCL of the MOB
compared with WT animals (Fig. 3H,I). We quantified the total
number of BrdU+ cells in coronal sections from the caudal OB (that
contains regions of both the MOB and the AOB) of E17.5 mice from
both genotypes and found no difference, suggesting that Tag1deficient OB projection neurons are redistributed rather than
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missing (Fig. 3J). The dorsoventral distribution of BrdU+ cells in
caudal OB cryosections from E17.5 WT and Tag1 −/− mice shows
that more Tag1-deficient MCs tend to remain in dorsal areas as
opposed to WT cells, which are equally distributed in dorsal and
ventral areas (Fig. 3K).
In order to locate E11.5-born projection neurons in the
developing OB, we followed them with BrdU IHC (injection at
E11.5) through the anteroposterior axis, including the rostral
(containing only the MOB) and caudal (containing both MOB
and AOB) regions. Coronal sections were subdivided into four
quadrants. The percentages of BrdU+/Tbr1+ cells were quantified
per OB quadrant and their distribution is shown in Fig. 4. At E14.5,
the dorsoventral distribution of E11.5-born cells is not significantly
different between Tag1 −/− and WT controls (Fig. 4A,A′,E).
However, at subsequent ages (E16.5, E17.5 and P0), a
dorsolateral preference (where the developing AOB is located) of
Tag1 −/− cells becomes evident, in contrast to the uniform
distribution in WT mice (Fig. 4B-D′). Quantification of the
number of BrdU+/Tbr1+ cells in dorsal versus ventral areas
confirmed their tendency to remain in dorsal areas in deficient
OBs (Fig. 4F-H). Finally, quantification of the cells in sections
containing exclusively the dorsal MOB (DMOB) or the AOB,
showed that the increase observed in the dorsolateral quadrant in P0
Tag1 −/− mice, results from their redistribution in the AOB (Fig. 4I).
In accordance to this finding, few GFP+ cells were found
DEVELOPMENT
Fig. 2. MCs are fewer in Tag1 −/− mice, whereas
TCs and OSNs are unaffected in number.
(A-C) Fluorescent ISH for Omp in E16.5 mouse
embryos showed no difference in the number of
mature OSNs in Tag1 −/− compared with WT
embryos (P>0.1, n=3). D-F. IHC for reelin in adult
mice showed no defect in the TC population in
Tag1 −/− animals (P>0.1, n=3). G-L. IHC in adult
(G-I) and P0 mice (J-L) for Tbx21. In the adult OB
(I), the number of Tbx21+ MCs is significantly
reduced in Tag1 +/− (14.2%, P=0.0058, n=6) and
Tag1 −/− mice (28.73%, P<0.001, n=6) compared
with age-matched WT animals. Quantification in P0
OBs (L), showed a significant decrease in Tag1 −/−
mice (24.8%, P=0.0012, n=4). To-Pro-3 (blue)
was used for nuclear staining. Error bars represent
s.e.m. **P<0.01, ***P<0.001. Scale bars: 150 μm in
A,B,G,H; 75 μm in D,E,J,K.
RESEARCH ARTICLE
Development (2015) 142, 4318-4328 doi:10.1242/dev.123943
mislocalized in the AOB of Tag1−/−;GFP mice (Fig. 4J-L). The
trajectories of OB projection neurons in the LOT and the PC display
no gross differences in Tag1 −/− mice compared with WT controls,
as indicated by IHC for the cell adhesion molecules L1 and
neuropilin 1(NP1, also known as Nrp1) (Fig. S5).
occupy all quadrants of the developing OB by E14.5, and that later
on, cells from the dorsolateral area need Tag1 to migrate towards the
developing MCL of the MOB.
In order to test this hypothesis, we performed organotypic
cultures of OB sections at E14.5 to examine the normal migration
pattern of projection neurons detected dorsolaterally, and to
investigate possible defects in this pattern in Tag1 −/− mice. DiI
tracing experiments (crystal placed in the dorsal part) showed that in
WT slices, cells migrate tangentially towards more rostral regions;
however in Tag1 −/− slices, only a few cells have left the dorsal part
after 2 days in culture, supporting our initial hypothesis (Fig. 5A).
We then set up a transplantation system (Fig. 5B), where the
transplants originated from the dorsal OB region (from lateral
slices) of Tag1 −/−;GFP or Tag1 +/+;GFP mice and the recipients
were WT or Tag1 −/− (Fig. 5Da-d). In another set of transplantation
experiments, donor cells were labeled by injecting pregnant donors
with BrdU at E11.5 (Fig. 5Da′-d′).
Migrating cells from dorsolateral OB (control experiment: donor,
Tag1 +/+;GFP; recipient, WT) are E11.5-born projection neurons
expressing Tag1, as shown by co-localization experiments for GFP
and Tbr1 or GFP and BrdU (Fig. 5C). All possible combinations of
transplants and recipients were performed, cultured for 3 days and
subjected to further sectioning and immunofluorescence for GFP
(Fig. 5Da-d) or BrdU (Fig. 5Da′-d′). We observed that Tag1 +/+
GFP+ or BrdU+ cells migrate normally on WT or Tag1 −/−
recipients, following a tangential pattern of migration towards
rostral and ventral areas (arrowheads in Fig. 5Da,a′,b,b′), suggesting
that Tag1 expression in the recipient is not a prerequisite for proper
migration. We combined transplants from BrdU-pulsed Tag1 −/− or
Tag1 −/−;GFP mice on WT or Tag1 −/− recipients (Fig. 5Dc,d,c′d′).
Donor cells in both cases display severe migration defects.
Specifically, only a few BrdU+ cells migrate from the transplant
area at short distances (quantified in Fig. 5E,F), whereas only a
small group of GFP+ cells migrate in the correct tangential direction
(# in Fig. 5Dc,d). These neurons might comprise later-born
populations, i.e. at E12.5 or E13.5. Therefore, Tag1 expression on
projection neurons from dorsolateral OB (born at E11.5) is
indispensable for their proper tangential migration and positioning
in the developing MCL of the MOB.
Tag1 is necessary for the tangential migration of E11.5-born
projection neurons from the dorsolateral OB
Tag1-deficient mice display severe alterations in their
olfactory ability and neuronal activation profile
MCs born at E11.5 are mislocalized in Tag1-deficient mice. We
hypothesize that through their radial migration, E11.5-born cells
In order to evaluate the olfactory ability of Tag1−/− mice, we used a
battery of behavioral tests. The buried food test is widely used for
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DEVELOPMENT
Fig. 3. Fewer E11.5-born cells reach the MCL of the MOB in Tag1 −/−
animals, through mislocalization. (A-C) OB coronal sections from newborn
Tag1loxP-GFP-loxP-DTA mice immunostained for GFP (green) and BrdU (red).
BrdU injections were performed at E10.5 (A), 11.5 (B) and 12.5 (C) in pregnant
animals. MCs born at E11.5 and 12.5 express Tag1 (arrowheads: double
stained cells), in contrast to E10.5-born MCs. (D,E) BrdU immunostaining
(green) in OB sections from P0 WT (D) and Tag1 −/− (E) mice, BrdU-pulsed at
E11.5 shows a reduction in Tag1 −/− MCL. (F-I). Quantification of BrdU+ cells in
OB sections from P0 (F,G) and E17.5 (H,I) mice (from rostral to caudal levels)
showed a reduction in the MOB of Tag1 −/− mice compared with WT controls
(at P0: 42.3%, P=0.0205, n=6; at E17.5: 19.48%, P=0.0416, n=5), and
increase in the AOB (at P0: 19.81%, P=0.0064, n=6; at E17.5: 37.6%,
P=0.0149, n=5). (J) The total number of BrdU+ cells was quantified exclusively
in caudal OB sections and no difference was detected (P>0.1, n=5) between
genotypes. (K) Graphical representation of the dorsoventral distribution of
BrdU+ cells in caudal OB cryosections from E17.5 WT and Tag1 −/− mice (n=3)
shows that whereas WT MCs are evenly distributed, in Tag1 −/− mice,
significantly more remain in dorsal areas. To-Pro-3 (blue) was used for nuclear
staining. Error bars represent s.e.m. Scale bars: 75 μm in A-C; 150 μm in D,E.
*P<0.05, **P<0.01.
RESEARCH ARTICLE
Development (2015) 142, 4318-4328 doi:10.1242/dev.123943
the evaluation of the ability of rodents to smell volatile odors. In this
test, Tag1−/− mice performed well, displaying an insignificant
increase in their latency to uncover the hidden food pellet, compared
with WT animals (Fig. 6A). In the social interaction test (Fig. 6B),
WT, Tag1+/− and Tag1−/− male mice were timed smelling an
anesthetized conspecific control animal on repeated trials. All
groups of animals progressively reduced the time spent exploring
the anesthetized conspecific (trials 1-4), indicative of efficient
perception and learning of its odor, although exploration time was
increased when the anesthetized animal was replaced with a new
one on trial 5. The olfactory memory task was performed 24 h later:
experimental animals were exposed to two anesthetized mice, the
familiar one (used for trials 1-4 of the learning phase) and a new
conspecific animal. Olfactory discrimination index (ODI), showed
that Tag1−/− mice display a severe defect in long-term social
memory (Fig. 6C). The observation of ODI on Tag1+/− animals is
important, as it appears significantly lower than WT but greater than
Tag1−/− mice (Fig. 6C). These mice display long-term memory
formation but perform worse in this social test that WT, suggesting a
correlation with the numerical defect on MCs.
In order to further elucidate the olfactory ability of Tag1-deficient
animals we used a more specific odor discrimination test, namely
the olfactory ‘habituation/dishabituation’ assay, using different
social (female odor and urine) and non-social [H2O, peanut butter
(PB) and 2-methylbutyric acid (2MB)] odors (Fig. 6D). The
repertoire of odorants contained both attractive (PB, social odors)
and repulsive (2MB) cues. In all odorants used, the odor preference
of Tag1−/− animals was significantly reduced compared with WT
controls (Fig. 6E; Movie 1). The most profound difference occurred
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in the attractive PB odor and in female social odors. These results
reveal the impaired olfactory ability of Tag1−/− mice regarding the
recognition and discrimination of odor cues. Tag1+/− mice display
defective odor recognition and discrimination but to a lesser extent
compared with Tag1−/− mice (Fig. 6E). To exclude the possibility
that our experimental Tag1−/− and Tag1+/− animals develop this
behavior as a result of anxiety-related defects we performed an
elevated plus maze test which revealed no difference between
genotypes (Fig. S6).
We next investigated whether the reduction in the number of MCs
of the MOB from Tag1 −/− mice results in alterations of the neuronal
activation profile that could account for these behavioral defects. No
difference was detected in the morphology and neuronal numbers of
PC between Tag1 −/− and WT mice (Fig. S7). Different groups of
male WT and Tag1 −/− mice were exposed to either PB for the
stimulation of the MOS, or female urine for the stimulation mainly
of the AOS (Fig. S8A). We used c-Fos (also known as Fos), encoded
by an immediate early gene, to monitor for neuronal activity as its
expression increases after synaptic stimulation (Datiche et al., 2001;
Roullet et al., 2005).
After PB stimulation, the number of c-Fos+ cells was quantified
in 4 different areas (dorsal, lateral, ventral and medial; Fig. 6F,
Fig. S8B-E) of the MCL (MOB) as well as in the PC of WT and
Tag1 −/− animals (Fig. 6G, Fig. S8F-G). There is a significant
reduction (30% and 40%, respectively) in the number of neurons
activated in the MOB and PC of Tag1 −/− mice compared with
controls (Fig. 6F,G). 74% of MCs that are born at E11.5 are
activated after PB stimulation, which represent 44% of total c-Fos+
cells (Fig. S8M). We propose that the olfaction defects of Tag1 −/−
DEVELOPMENT
Fig. 4. Tracking the location of Ε11.5-born MCs in the developing OB. (A-D′) Radial graphs showing the percentages of BrdU+/ Tbr1+ MCs in different
quadrants of the entire OB. BrdU was injected at E11.5 in WT (A-D) and Tag1 −/− (A′-D′) mice and the analysis was performed at E14.5 (P=0.5037, n=3, A,A′),
E16.5 (P=0.0208, n=3, B,B′), E17.5 (P=0.0074, n=3, C,C′) and P0 (P=0.0034, n=4, D,D′). (E-H) Graphical representation of the dorsoventral distribution of
E11.5-born MCs at E14.5-P0. (I) Percentage of double positive cells that are found in dorsal MOB (DMOB) in comparison with the AOB area in P0 animals.
(J-L) Using Tag1 loxP-GFP-loxP-DTA mice, we found that GFP+ cells are increased inside the AOB of Tag1 −/− mice. GFP+ cells are indicated on the border
(arrowheads) or inside the AOB (arrows). Error bars represent s.e.m. *P<0.05, **P<0.01. DM, dorsomedial; DL, dorsolateral; VM, ventromedial; VL, ventrolateral.
RESEARCH ARTICLE
Development (2015) 142, 4318-4328 doi:10.1242/dev.123943
mice could result from the reduced output of MCs towards the
olfactory cortex, after exposure to volatile odor stimuli. No
difference between genotypes was found in the c-Fos profile in
the OE, suggesting that activity of the initial station of the
odorant information route is not affected (Fig. S8J-L).
Furthermore, no difference was detected in the activation profile
or the total number of GABA+ cells in the granule cell layer in
Tag1 −/− mice (Fig. S8N,O).
In order to activate the AOB of adult male mice we used a female
urine cue, containing non-volatile signals (in addition to volatile
molecules), which are detected by the vomeronasal organ. IHC for
c-Fos in adult mice showed that there is no significant difference in
the number of activated neurons in Tag1 −/− animals, suggesting
intact AOB responsiveness after stimulation (Fig. 6H, Fig. S8H,I).
DISCUSSION
Despite progress on the elucidation of the olfactory receptor map
(Bozza et al., 2009; Mori et al., 1999; Mori et al., 2006), little is
known about the organization, function and circuit integration of the
OB projection neurons. The aim of this study is to shed light on the
organization of projection neurons inside the OB and show that
alterations in this organization affect olfactory function in mice
lacking Tag1 expression.
Tag1 is strongly expressed by immature as well as migrating MCs
and TCs of the developing OB. Furthermore, it is expressed by a
subset of OSNs, although its absence in Tag1 −/− mice does not
affect the number of projection to the glomerular layer of the OB.
We detected a significant reduction in the number of mature MCs in
adult Tag1 −/− mice, whereas TCs were unaffected. The projections
of MCs into the LOT of Tag1 −/− mice towards the olfactory cortex
led to neither defects in their fasciculation nor a reduction in the
thickness of the axonal tract, suggesting that the redistributed MCs
are still able to project normally. MCs are classified according to
their birth dates as either early-born (at E10 or earlier) and late-born
(at E12 or later). These are localized in the dorsomedial and
ventrolateral MCL, respectively, whereas MCs born at E11.5 are
uniformly distributed inside the MCL (Imamura et al., 2011). We
showed that populations of late-born and intermediate MCs born
at E11.5 express Tag1. E11.5-born MCs undergo proper radial
migration and thus are uniformly distributed by E14.5 in Tag1−/−
mice, but are unable to change from a radial to a tangential-like route
and subsequently migrate towards the anterior and ventral areas of
the developing OB. Transplantation experiments suggest a cell
autonomous defect.
Our results are in line with a proposed role of Tag1 in facilitating
tangential migration in other neuronal systems, such as the
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DEVELOPMENT
Fig. 5. Tag1+ OB projection neurons migrate tangentially from the dorsal part and their migration is defective in Tag1 −/− mice. (A) Organotypic cultures of
sagittal sections from E14.5 brains. A DiI crystal was placed in the dorsocaudal part of lateral OB vibratome sections as shown in the scheme, and the
sections were cultured for two days. (B) Transplants were generated on sagittal E14.5 sections as shown in the scheme, with donor cells expressing GFP under
the Tag1 gene promoter or BrdU-pulsed at E11.5. (C) WT GFP+ and BrdU+ (BrdU injection at E11.5) cells were transplanted into WT recipients and
immunostained for GFP and Tbr1 or GFP and BrdU. Migrating cells (GFP+) are projection neurons (co-localization with Tbr1, upper images) and are born at E11.5
(co-localization with BrdU, lower images). (D) All possible combinations of transplants are presented immunostained for GFP or BrdU (shown in green). WT GFP+
or BrdU+ cells migrated on either WT or Tag1 −/− recipients, with some cells reaching the anterior and ventral part of the section (arrowheads in a,a′,b,b′), but only a
few Tag1 −/−;GFP+ cells migrated on either WT or Tag1 −/− recipients (# in c,d). Asterisks in a,a′,b,c′,d indicate a migratory stream, occasionally detected. To-Pro-3
(blue) was used for nuclear staining. Dashed white lines represent the transplant limits. (E,F) Quantification of the average number of migrating neurons and the
average distance travelled. n=7-9, *P<0.05, **P<0.01, ***P<0.001. Error bars represent s.e.m. Scale bars: 300 μm in A, 150 μm in C,D.
RESEARCH ARTICLE
Development (2015) 142, 4318-4328 doi:10.1242/dev.123943
precerebellar nuclei (Kyriakopoulou et al., 2002; Denaxa et al.,
2005). There, Tag1 mediates migration through chain interaction
among Tag1-expressing axons and soma-axonal appositions
between Tag1+ cells and Tag1− axonal scaffold. Imamura et al.
(2011) showed that E12.5-born MCs migrate tangentially in contact
with Ocam+ preexisting axons of the LOT. Tag1 protein is detected
on the cell body of migrating MCs until E16.5, when their tangential
migration is almost complete, suggesting that this migration might
occur through a soma-axonal apposition mechanism between MC
somata and preceding axonal tracts.
It is established that Tag1 exerts its function through either
homophilic and/or heterophilic interactions, although our
transplantation data indicates that a trans homophilic interaction is
improbable in our system (Karagogeos, 2003; Traka et al., 2003;
Lieberoth et al., 2009; Ma et al., 2008). Candidate molecules for
heterophilic interactions could be the adhesion molecules L1 and
4324
Ocam, which are also expressed developmentally by the axons of
OB projection neurons (Treloar et al., 2003; Inaki et al., 2004).
Do the molecular deficits present in the MOS of Tag1−/− mice
account for any behavioral defects related to olfaction? We have
shown that homozygous mutants display a series of behavioral
alterations such as learning and memory defects as well as motor
coordination and balance abnormalities, resulting from the
molecular disorganization of myelinated fibers (Traka et al., 2003;
Savvaki et al., 2008, 2010). When the juxtaparanodal domain
organization of myelinated fibers was rescued in vivo, learning and
memory defects that are dependent on hippocampal function were
also rescued (Savvaki et al., 2010). Our present analysis
reveals that Tag1 −/− mice are able to smell volatile and nonvolatile odors in the buried food and social interaction tests
respectively, but show a profound disruption in odor discrimination
and long-term social memory formation. Aged individuals and
DEVELOPMENT
Fig. 6. Tag1-deficient mice display altered olfaction and neuronal activation profile. (A) Time taken for WT and Tag1 −/− mice to locate food in the buried food
test, n=9. (B) Social interaction times during the olfactory learning task in WT, Tag1 +/− and Tag1−/− mice. One-way ANOVA with repeated measurements (trials),
n=4. On trial 5, we used a new conspecific animal. (C) Social memory test. The olfactory discrimination index (ODI) was calculated. Tag1 −/− mice show
ODI close to 0 (P=0.0054, n=4). Tag1 +/− animals present an intermediate ODI between WT and Tag1−/− (P=0.0138 compared with WT, n=4; P=0.0407 compared
with Tag1−/−, n=4). (D) Habituation/dishabituation test scheme. (E) Habituation/dishabituation test with social and non-social odors reveals an altered ability of
Tag1 −/− and Tag1 +/− mice to detect volatile odors. For each odor tested, one-way ANOVA with repeated measures (trials), trial×genotype interaction, P<0.01, n=9
for WT and Tag1 −/−, n=8 for Tag1 +/−. Red asterisks represent statistically significant difference between WT and KO, Green asterisks between WT and HET and #
between HET and KO. Colored dashed lines and bars denote three trials for a specific odor. S.O., social odor. (F-H) Neuronal activation was measured in WT and
Tag1−/− mice, using c-Fos antibody after exposure of the animals to peanut butter. Reduced number of activated neurons was detected in the MCL (30%,
P=0.0291, n=4) and the PC (40.10%, P=0.0028, n=4) of adult Tag1 −/− mice. No difference was detected in the AOB after nasal exposure to female urine (P=0.5,
n=4). Error bars represent s.e.m. *P<0.05, **P<0.01, ***P<0.001.
RESEARCH ARTICLE
Fig. 7. Model of olfactory bulb organization and output to olfactory
cortices in WT and Tag1 −/− mice. The absence of Tag1 in Tag1 −/− mice
results in a reduced number of MCs found in the MCL of the main olfactory bulb
which send reduced output to the piriform corteх. Subsequently, reduced
activation of the piriform cortex significantly affects olfactory behavior. PC,
Piriform cortex.
Tag1−/− mice, subtle alterations in OE function, i.e. in its
electrophysiological properties, cannot be excluded.
This is the first study that proposes a molecular cue involved in
the organization and development of projection neuron circuitry in
the OB. Tag1 is a key molecule that coordinates the integration of
E11.5-born MCs inside the developing MC map of the MOB. Its
absence results in aberrant decoding of odorant information towards
olfactory cortices, ending up in a disturbed behavioral phenotype
regarding olfaction (Fig. 7).
MATERIALS AND METHODS
Animals
The generation of Tag1−/− mice has been described (Fukamauchi et al.,
2001; Traka et al., 2003; Savvaki et al., 2008). All mice were kept as
heterozygote breeding pairs and the genotypes were confirmed by PCR.
Genetically modified Tag1 loxP-GFP-loxP-DTA mice were generated using BAC
technology. These mice express GFP under the Tag1 promoter without
affecting endogenous expression. They were crossed with Tag1+/− and
Tag1−/− mice in order to obtain the genotypes of interest (Tag1 +/+;GFP,
Tag1 −/−;GFP). E0.5: day of the vaginal plug. All mice were of C57BL6/
SV129 background. Housing and animal procedures used were according to
the European Union policy (Directive 86/609/EEC) and institutionally
approved protocols.
Immunohistochemistry
Immunohistochemistry (IHC) was performed on cryosections as described
(Vidaki et al., 2012) with the exception of adult OB cryosections (14 μmthick), which were post-fixed in ice-cold acetone for 10 min, blocked (in 5%
BSA in 0.1 M PBS) and incubated with primary and secondary antibodies in
5% BSA (Sigma-Aldrich) 0.5% Triton-X in 0.1 M PBS. Antibodies used:
rabbit polyclonal anti-Tbr1 (T-box brain 1, cat. no. Ab31940, Abcam,
1:600), rat polyclonal anti-GFP (cat. no. 04404-84, Nacalai Tesque,
1:1000), rabbit polyclonal anti-Tag1 (TG3, 1:1000, Traka et al., 2003),
rabbit anti-Tbx21 (T-box transcription factor, kind gift from Y. Yoshihara,
1:10,000) rabbit polyclonal OCAM (kind gift from Y. Yoshihara, 1:1000),
4325
DEVELOPMENT
patients with neurodegenerative diseases have been reported to
display hyposmia as well as impaired odor discrimination and
identification, with underlying pathophysiological alterations
throughout the olfactory system (Card et al., 1988; Attems et al.,
2014). In a mouse model of Alzheimer’s disease, denervation of the
OB resulted in reduced amyloid plaque load in the OB, the
neocortex and the hippocampus (Bibari et al., 2013). Inside the OB,
amyloid precursor protein is predominantly expressed by MCs
(Card et al., 1988). These studies emphasize the importance of the
olfactory system and the crucial role that main projection neurons of
the OB could play in the affected olfaction of patients with
neurodegenerative diseases.
It is well known that the MOS and the AOS participate in social
behaviors such as sex recognition of conspecifics, by detecting
volatile and non-volatile molecules from urine or other body
odorants, respectively (O’Connell and Meredith, 1984; Pankevich
et al., 2004). The volatile sex-specific cues detected mainly by the
MOS guide the animal towards the odorant source. After direct
interaction with the cue, the AOS is activated, subsequently
triggering different behavioral and neuroendocrine responses
(Humphries et al., 1999; Pankevich et al., 2004; Hurst, 2009).
Olfactory ‘habituation/dishabituation’ odor discrimination assays
showed that Tag1 −/− animals were unable to discriminate among
volatile stimuli (PB, social odor and 2MB). When we used female
urine (containing also non-volatile cues), Tag1 −/− mice performed
better than with the previous stimuli, but still significantly worse
than the control animals. This suggests that the AOS is at least
partially functional, but the defects in MOS render deficient mice
unable to discriminate cues. Tag1+/− mice display altered odor
discrimination ability in this test, but they perform better than
Tag1−/− animals, supporting the gene dose-dependent effect. Intact
AOS activity is further supported by our results on neuronal
activation. The social interaction test revealed that Tag1−/− mice are
able to investigate social stimuli and develop short-term social
memory, but display severe defects in long-term social memory.
This deficit can be attributed to both hippocampal and olfactory
discrimination defects. Our data from Tag1 +/− mice, where
myelinated fibers are not affected in the hippocampus and other
brain regions (Savvaki et al., 2008) but long-term social memory
is altered, support the contribution of defective olfactory
discrimination in this genotype. Noack et al. (2010) showed that
long-term social recognition in mice includes the neuronal
activation of both the MOB and the AOB, contrary to rats, where
the AOB is mainly activated. Furthermore, they showed that the
non-volatile fraction (received by AOS) is sufficient for the
formation of short-term social memory, whereas in the case of
long-term social memory, both the volatile and non-volatile
fractions are required.
Our results propose that MCs born at E11.5 are of major
importance for the proper function of the OB. Our hypothesis is
reinforced by a recent study showing that MCs are heterogeneous in
terms of their biophysical properties and that even a small imbalance
in this population could significantly influence OB function (Kollo
et al., 2014). These researchers showed that approximately one third
of MCs and TCs are found in a ‘silent’ state, with very low baseline
activity. However, these cells, in contrast to ‘highly active’
projection neurons, can vigorously respond to odorant stimulation.
Therefore, the 25% reduction of the otherwise uniformly distributed
MC population, born at E11.5, could be correlated with the severe
defects in olfactory recognition and discrimination that are observed
in Tag1 −/− mice. Although the gross morphology of the OE as
well as the organization of the glomerular map is not altered in
Development (2015) 142, 4318-4328 doi:10.1242/dev.123943
RESEARCH ARTICLE
BrdU injection and immunodetection
BrdU injection and immunodetection were performed as previously
described (Vidaki et al., 2012).
OB transplants and immunofluorescence
Brains from E14.5 embryos were dissected in L15 medium (Gibco)
containing 100 μ/ml pen/strep (Biosera). Cortices with OBs were embedded
in 3% low melting agarose (LMA, Lonza) in L15. Sagittal vibratome
sections 250 μm thick were placed on BIOPORE membranes (Millipore) in
DMEM/ F12A (DMEM/F12, 0.6% glucose, 5% FBS, 100 μ/ml pen/strep,
1× Glutamax, 1% N2 supplement). Sections were incubated at 37°C, 5%
CO2 for 1 h. Afterwards, the transplant was excised from the donor OB and
carefully placed on the recipient (after removing the appropriate part). The
medium was replaced by fresh Neurobasal/B27 (Neurobasal, 0.6% glucose,
100 μ/ml pen/strep, 1× Glutamax, 2% B27 supplement) and sections were
incubated for 3 days at 37°C, 5% CO2. For immunofluorescence, sections
were fixed for 1 h in 4% PFA and re-sectioned (60 μm) in 3% LMA in 0.1 M
PBS, washed in 0.1 M PBS containing 0.1% Triton-X (1× PBT) and
blocked in 1% FBS in 1× PBT for 1 h at room temperature. Finally, sections
were incubated with primary and the secondary antibodies in blocking
solution, washed and subbed in 0.2% (w/v) porcine skin gelatin in 50 mM
Tris pH 7.5. Slides were mounted with MOWIOL and observed in a Leica
TS2 confocal microscope. Antibodies used: rat polyclonal anti-GFP
(cat. no. 04404-84, Nacalai Tesque, 1:1000), rabbit polyclonal anti-GFP
(cat. no. 721, Minotech Biotechnology, 1:50,000), rabbit polyclonal antiTbr-1 (cat. no. Ab31940, Abcam, 1:600), rat anti-BrdU (cat. no.
OBT0030CX, IgG, AbD Serotec, 1:1000) and fluorochrome-labeled
secondary antibodies Alexa Fluor 488 and 555 (Molecular Probes, 1:800).
In situ hybridization (ISH) and fluorescent ISH
ISH was performed on coronal cryosections as described (Denaxa et al.,
2001; Ishii et al., 2004). Antisense probes for mouse Tag1 (Denaxa et al.,
2001), Omp, Gap43, Mash1 (kind gifts from P. Mombaerts) and Ngn1 (kind
gift from F. Guillemot) were used.
Behavioral analysis
Buried food test
Two-month old (mo) male WT and Tag1 −/− mice (n=6) were assayed for
their ability to locate a hidden, highly palatable food piece (cheese chip)
underneath the cage bedding as described (Yang and Crawley, 2009) with
minor modifications: a cheese chip was hidden ∼1 cm beneath the cage
bedding in a random corner of the experimental cage. We consider that the
animal had found the cheese chip when it held it in its front paws and began
to eat it.
Social interaction test
Social interaction test was performed as previously reported with minor
modifications (Kogan et al., 2000). The olfactory discrimination index
(ODI) indicates the memory ability of the animals to recall a previously
experienced olfactory stimulus (see also supplementary materials and
methods).
Neuronal activity experiment, c-Fos labeling and analysis
MOS, AOS activation protocol
Two-month old male mice were used. For MOS activation, a stimulus of
10% w/v PB (dissolved in odorless paraffin oil) or H2O was used. Three to
four hours before the test initiation, each animal was transferred in a clean
cage (with filter cap) without food, H2O or bedding under the fume hood.
Afterwards, the PB odor was introduced into the cage through two cotton tip
applicators (each containing 150 μl of PB) and mice were left to smell the
stimulus for 90 min. Then, experimental animals were deeply anesthetized,
perfused with 4% PFA and brains were dissected. For the AOS we followed
the protocol from Pierman et al. (2008) and Veyrac et al. (2011) with minor
modifications. After the habituation period, 30 μl of female urine (from a
pool of female urine) was applied inside the nose of the subjects (or H2O in
controls) and mice were then placed back in the filter cage for 90 min. The
following steps were the same as in MOS activation protocol.
c-Fos labeling
A modified protocol was used for the IHC of c-Fos protein in adult OB and
PC cryosections (Sundquist and Nisenbaum, 2005); see also supplementary
materials and methods. For the c-Fos/BrdU co-labeling the c-Fos protocol
preceded the BrdU protocol.
Image analysis and statistics
For the MOS activation analysis, serial sections were obtained at 10× and
40× magnification. At 40× magnification representative images from the
MOB were acquired with the same settings for all animals. For PC and
granule cell analysis, four images from serial sections (20× magnification)
were used, spanning anterior to more posterior regions. For AOS activation
analysis, 20× magnification confocal images were acquired and used from
two regions of the AOB (anterior and posterior). In all cases, quantification
was performed with ImageJ (National Institutes of Health).
Statistics/radial statistics analysis
Student’s t-test and one-way ANOVA were used for the statistical analysis
by GraphPad Prism version 5.00. Regarding radial statistics analysis, for all
developmental stages we obtained sections representative of the entire OB
tissue through the anteroposterior (AP) axis. At all developmental stages
tested, 3-4 regions were used through the AP axis in both genotypes. Each
section was divided into four different anatomical quadrants (dorsolateral,
ventrolateral, ventromedial and dorsomedial). Then, double positive (Tbr1/
BrdU) cells were manually counted and the total number of cells per
quadrant as well as the total percentage of cells per quadrant was calculated
for each section and for each age. Radial statistics graphs were generated on
Grapher 8 (v8.7, Golden Software), using the average percentage of cells per
quadrant (in all sections) for all animals of each genotype. Statistical
analysis of dorsoventral positioning was performed using the mean
percentages of cells found on dorsal (DL, DM) and ventral (VL, VM)
quadrants in all sections of the OB for each animal and genotype (unpaired
Student’s t-test was performed using GraphPad Prism version 5.00).
Acknowledgements
The authors are grateful to Drs Sonia Garel (IBENS-INSERM-CNRS, Paris, France),
Yoshihiro Yoshihara (RIKEN Brain Science Institute, Japan), H. Sakano (The
University of Tokyo, Japan) and Francois Guillemot (NIMR-MRC, London) for
providing reagents. We would also like to thank Drs Peter Mombaerts and Markella
Katidou (Max Planck Research Unit for Neurogenetics, Frankfurt) for training MS in
fluorescent ISH and providing reagents. We also thank Drs K. Kleopa, M. Strigini,
L. Zoupi for their comments on the manuscript.
Competing interests
Habituation/dishabituation test
The test was performed as previously described with minor modifications
(Basic Protocol 2) (Yang and Crawley, 2009); see also supplementary
materials and methods.
Elevated plus maze
The test was performed as previously described (Stamatakis et al., 2015).
4326
The authors declare no competing or financial interests.
Author contributions
S.M. and B.G.G. contributed equally to this work. Both of them designed and
performed experiments, analyzed data and wrote the paper. S.M. also supervised
the work. S.A. performed some behavioral experiments and contributed to the
loxP-GFP-loxP-DTA
mouse
analysis of the data. V.M. designed and generated the Tag1
line. K.D. supervised the work and wrote the paper.
DEVELOPMENT
guinea pig a-mOR-EG (kind gift fromY. Yoshihara, 1:1000), guinea pig
a-OR-I7 (kind gift from Y. Yoshihara, 1:5000), rabbit a-Kirrel2 [ protocol
described in Serizawa et al. (2006), a kind gift from H. Sakano, 1:1000] and
fluorochrome-labeled secondary antibodies Alexa Fluor 488 and 555 (cat.
no. A11034, A21429, A21435, Molecular Probes, 1:800). To-Pro 3 iodide
(Invitrogen) was used for the visualization of the nuclei. Hematoxylin/Eosin
and Nissl staining was used for morphological characterization.
Development (2015) 142, 4318-4328 doi:10.1242/dev.123943
Funding
This work was supported by the European Commission FP7 programme
‘Translational Potential’ [contract number 285948], InnovCrete [316223], ARISTEIA
I [Project 593 MyelinTag] and by Institute of Molecular Biology and Biotechnology
intramural grants. B.G.G. has been the recipient of the Manasaki Fellowship and
Medical School Fellowship of the University of Crete. V.M. is currently a postdoctoral
fellow at the Koch Institute, MIT, Cambridge, MA, USA.
Supplementary information
Supplementary information available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.123943/-/DC1
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