BIG-2 Mediates Olfactory Axon
Convergence to Target Glomeruli
Tomomi Kaneko-Goto,1Sei-ichi Yoshihara,1,3Haruko Miyazaki,1,4and Yoshihiro Yoshihara1,2,*
1Laboratory for Neurobiology of Synapse, RIKEN Brain Science Institute, Saitama 351-0198, Japan
2Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Osaka 560-0082, Japan
3Present address: Department of Molecular Biology for Neural Systems, Research Institute for Frontier Medicine,
Nara Medical University, Nara 634-8521, Japan.
4Present address: Laboratory for Structural Neuropathology, RIKEN Brain Science Institute, Saitama 351-0198, Japan.
Olfactory sensory neurons expressing a given odor-
ant receptor converge axons onto a few topographi-
cally fixed glomeruli in the olfactory bulb, leading to
establishment of the odor map. Here, we report that
BIG-2/contactin-4, an axonal glycoprotein belonging
to the immunoglobulin superfamily, is expressed in
a subpopulation of mouse olfactory sensory neu-
rons. A mosaic pattern of glomerular arrangement
is observed with strongly BIG-2-positive, weakly
positive, and negative axon terminals in the olfactory
bulb, which is overlapping but not identical with
those ofKirrel2 and ephrin-A5.There is aclose corre-
lation between the BIG-2 expression level and the
odorant receptor choice in individual sensory neu-
rons. In BIG-2-deficient mice, olfactory sensory neu-
rons expressing a given odorant receptor frequently
innervate multiple glomeruli at ectopic locations.
These results suggest that BIG-2 is one of the axon
guidance molecules crucial for the formation and
maintenance of functional odor map in the olfactory
The olfactory system furnishes sophisticated molecular and
cellular mechanisms for reception, discrimination, and internal
representation of information carried by an immense number of
odor molecules in the external world. To achieve this feat, olfac-
tory sensory neurons (OSNs) in the olfactory epithelium (OE) are
destined to obey two basic principles during their unique differ-
for expression from a repertoire of ?1000 genes in mice, being
tuned to a range of odor ligands with shared structural features
that can bind and activate the expressed OR (Chess et al.,
1994; Malnic et al., 1999; Serizawa et al., 2003; Lewcock and
Reed, 2004; Shykind et al., 2004). Second, OSNs expressing
a given OR project and converge their axons onto a few topo-
graphically fixed glomeruli among ?2000 glomeruli spatially
arranged on the surface of the olfactory bulb (OB), resulting in
establishment of the ‘‘odor map’’ in OB (Vassar et al., 1994;
Ressler et al., 1994; Mombaerts et al., 1996; Mori et al., 1999,
Thus, we currently have a wealth of knowledge on the pattern
of olfactory axon wiring and the representation of odor informa-
tion on OB odor map. So far, several guidance systems have
been identified that play distinct roles in step-wise processes
including Cxcl12/Cxcr4, Robo/Slit, neuropilin/semaphorin, Eph/
ephrin, and Kirrel2/3 (Miyasaka et al., 2005, 2007; Schwarting
et al., 2000; Walz et al., 2002; Taniguchi et al., 2003; Imai et al.,
2006; Cutforth et al., 2003; Serizawa et al., 2006). However,
the precise molecular mechanisms underlying the formation
and maintenance of glomerular targeting still remain to be
BIG-2 (contactin-4) is a glycosylphosphatidylinositol (GPI)-
anchored axonal glycoprotein with six immunoglobulin (Ig)-like
domains and four fibronectin-type III (FnIII) repeats (Figure 1A),
belonging to the contactin subgroup of the Ig superfamily (Yosh-
ihara et al., 1995), also referred to contactin-4 (Zeng et al., 2002;
Hansford et al., 2003). BIG-2 shows a unique expression pattern
in neuron type- and developmental stage-specific manners in
various regions of the nervous system (Yoshihara et al., 1995).
In the present study, we investigated the expression of BIG-2 in
the mouse olfactory system and found the mosaic pattern of
BIG-2 protein expression among glomeruli. The expression level
Furthermore, in BIG-2-deficient mice, olfactory axons frequently
role of BIG-2 in the establishment of precise odor map in OB.
Expression of BIG-2 in a Subset of OSNs
In the course of immunohistochemical staining of mouse brain
sections with antibodies against various cell recognition and
axon guidance molecules, we noticed the strong expression of
BIG-2 protein in the olfactory nerve layer and glomerular layer
of OB. This preliminary finding prompted us to investigate de-
tailed localization and physiological function of BIG-2 in the
primary olfactory system.
In situ hybridization analysis of coronal sections of 6-week-old
mouse heads at different anteroposterior levels showed that
834 Neuron 57, 834–846, March 27, 2008 ª2008 Elsevier Inc.
in the vomeronasal organ (Figures 1B–1D). A higher magnifica-
tion of the OE section revealed that BIG-2 mRNA was present
cells (Figure 1E). BIG-2-negative OSNs (black asterisks in Fig-
ure 1E) were intermingled with BIG-2-positive OSNs (white
asterisks in Figure 1E) in OE.
nohistochemistry with anti-BIG-2 antibody. BIG-2 protein was
abundantly present in axons of OSNs but was almost absent
from their dendrites and cell bodies (Figure 1F), which is a char-
acteristic pattern of subcellular localization for most of GPI-
anchored neuronal proteins (Faivre-Sarrailh and Rougon, 1997).
BIG-2-positive axon bundles were observed throughout OE,
similar to NCAM-positive axons (Figures 1F–1H). A higher mag-
nification of triple-labeled axon bundles with antibodies against
BIG-2, NCAM, and OCAM revealed that BIG-2-positive fibers
(Figures 1I and 1L–1N) constitute a subpopulation, but not all,
of axon bundles of OSNs, which partially overlaps but is distinct
from a population of NCAM-positive fibers (Figures 1J, 1L, and
1N). The expression pattern of BIG-2 did not correspond to the
olfactory zones whose boundary (between zone 1 and 2) can
be evidently delineated by OCAM-negative and -positive axons
(Figures 1K, 1M, and 1N; Yoshihara et al., 1997). These results
indicate that BIG-2 is expressed by a subset of OSNs with no
relation to the zonal organization of OE.
Mosaic Pattern of BIG-2 Protein Expression
among OB Glomeruli
To investigate the glomerular targeting patterns of BIG-2-posi-
tive and -negative axons, we performed immunohistochemical
analysis of BIG-2 expression in OB sections from 6-week-old
mice. A parasagittal section containing the glomerular sheet
of the medial OB showed that individual glomeruli are labeled
at different intensities with anti-BIG-2 antibody. Strongly BIG-
2-positive glomeruli were intermingled with weakly BIG-2-
positive and BIG-2-negative glomeruli on the surface of OB,
whereas almost all the glomeruli displayed a similar level of
NCAM expression (Figures 2A–2C). The distribution pattern of
glomeruli positive for BIG-2 was completely different from
those for OCAM and neuropilin-1 (NP-1) (Figures 2D–2K), indi-
cating that BIG-2 expression does not relate to the dorsoventral
Figure 1. Expression of BIG-2 mRNA and Protein in a Subset of OSNs
(A) A schematic diagram depicting the structure of BIG-2 protein. BIG-2 consists of N-terminal signal peptide (SP), six Ig-like domains (Roman numerals), four
FnIII-like domains (Arabic numerals), and C-terminal GPI tail. S-S: a disulfide bond in each Ig-like domain.
a subset of OSNs. White asterisks: OSNs with a high level of BIG-2 mRNA expression. Black asterisks: OSNs devoid of BIG-2 mRNA.
(F–H)DoublelabelingofacoronalOEsectionwithantibodiesagainstBIG-2andNCAM.BIG-2 protein(F)isexpressedinOSNaxonsextending fromOEaswellas
NCAM (G). (H) A merged image showing BIG-2 (red) and NCAM (green).
(I–N) Triple labeling of OSN axon bundles projecting from OE to OB with antibodies against BIG-2, NCAM, and OCAM. BIG-2 protein (I) is present in a subset of
OSN axons that are overlapping but different from NCAM-positive (J) and OCAM-positive axons (K). (L–N) Merged images of BIG-2 (red), NCAM (green), and
OCAM (blue) signals.
Scale bars: (B–D) and (F–H) 1 mm, (E) 25 mm, (I–N) 100 mm.
BIG-2 Mediates Olfactory Axon Targeting
Neuron 57, 834–846, March 27, 2008 ª2008 Elsevier Inc. 835
We thank K. Mori for continuous support and encouragement; H. Sakano for
gifts of anti-Kirrel2 antibody and H-MOR28-ires-tauECFP transgenic mice;
H. Breer for a gift of anti-mOR256-17 antibody; F.L. Margolis for a gift of
anti-OMP antibody; T. Kaneko for a gift of anti-VGluT2 antibody; Y.F. Sasaki
for help in production of anti-BIG-2 antibody; S. Mitsui for help in production
of anti-protocadherin-21 antibody; K. Kubota and M. Kawasaki for help in
construction of BIG-2 targeting vector; Y. Furutani for help in production of
recombinant proteins; BSI Research Resource Center (RRC) for help in gener-
ation of BIG-2-deficient mice; N. Miyasaka for comments on the manuscript;
and members of the Yoshihara lab for valuable discussions. This work was
supported in part by a Grant-in-Aid for Scientific Research (B) and a Grant-
in-Aid for Scientific Research on Priority Area (Cellular Sensor) to Y.Y. from
the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Received: August 10, 2007
Revised: December 6, 2007
Accepted: January 18, 2008
Published: March 26, 2008
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BIG-2 Mediates Olfactory Axon Targeting
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