RESEARCH ARTICLE 2783
Development 139, 2783-2791 (2012) doi:10.1242/dev.077008
© 2012. Published by The Company of Biologists Ltd
The wiring of Grueneberg ganglion axons is dependent on
neuropilin 1
Tomohiko Matsuo, Daniel Aharony Rossier, Chenda Kan and Ivan Rodriguez*
SUMMARY
The Grueneberg ganglion is a specialized olfactory sensor. In mice, its activation induces freezing behavior. The topographical map
corresponding to the central projections of its sensory axons is poorly defined, as well as the guidance molecules involved in its
establishment. We took a transgenic approach to label exclusively Grueneberg sensory neurons and their axonal projections. We
observed that a stereotyped convergence map in a series of coalescent neuropil-rich structures is already present at birth. These
structures are part of a peculiar and complex neuronal circuit, composed of a chain of glomeruli organized in a necklace pattern
that entirely surrounds the trunk of the olfactory bulb. We found that the necklace chain is composed of two different sets of
glomeruli: one exclusively innervated by Grueneberg ganglion neurons, the other by axonal inputs from the main olfactory
neuroepithelium. Combining the transgenic Grueneberg reporter mouse with a conditional null genetic approach, we then show
that the axonal wiring of Grueneberg neurons is dependent on neuropilin 1 expression. Neuropilin 1-deficient Grueneberg axonal
projections lose their strict and characteristic avoidance of vomeronasal glomeruli, glomeruli that are innervated by secondary
neurons expressing the repulsive guidance cue and main neuropilin 1 ligand Sema3a. Taken together, our observations represent a
first step in the understanding of the circuitry and the coding strategy used by the Grueneberg system.
INTRODUCTION
Multiple and specialized sensory systems have been selected
during the evolution of vertebrates. Among these sensors, olfaction
represents a major tool for many species. In most mammals, the
olfactory system is divided into at least three physically and
functionally separate sensory structures. These consist of the main
olfactory neuroepithelium, the vomeronasal organ and the
Grueneberg ganglion. This last structure is located at the tip of the
nose, in the vestibule of the anterior nasal cavity, and is composed
of a few hundred sensory neurons (Gruneberg, 1973).
The function of the Grueneberg ganglion is apparently
multimodal, as it is responsive to both thermo- and chemostimuli
(Mamasuew et al., 2008; Chao et al., 2010; Schmid et al., 2010;
Mamasuew et al., 2011). The structure also responds to volatile
molecules produced by stressed mice, an activation that leads to
a freezing behavior in the recipient animals (Brechbuhl et al.,
2008).
Sensory information is spatially encoded in the brain of
vertebrates. This is also true for olfactory information, which is
relayed to the brain through sensory axonal projections that
coalesce into anatomical structures called glomeruli. Axonal
projection maps in the olfactory bulb are characteristic of each type
of olfactory sensory neuron. The axonal projections of the
Grueneberg ganglion have only recently been associated with the
olfactory system (Fleischer et al., 2006a; Fuss et al., 2005; Koos
and Fraser, 2005; Roppolo et al., 2006; Storan and Key, 2006).
They converge in the olfactory bulb and are part of a group of
Department of Genetics and Evolution and National Research Center Frontiers in
Genetics, University of Geneva, 1211 Geneva 4, Switzerland.
*Author for correspondence ([email protected])
Accepted 14 May 2012
glomeruli called the necklace glomeruli (Shinoda et al., 1989).
These constitute a very peculiar and still poorly defined set of
glomeruli, which are linked with each other in a chain-like narrow
band that runs between the main and the accessory olfactory bulbs
and that surrounds the entire trunk of the olfactory bulb (Fig. 1A).
This necklace-shaped group of glomeruli is also innervated by
sensory neurons located in the main olfactory system, which detect
a variety of semiochemicals (Hu et al., 2007; Leinders-Zufall et al.,
2007; Munger et al., 2010). Whether specific necklace glomeruli
are co-innervated by Grueneberg and main olfactory projections is
unknown.
The establishment of an olfactory topographic map in the bulb
results from the integration by axonal fibers of various guidance
cues. The immense majority of studies have focused their attention
on the molecular signals allowing sensory neurons to navigate
toward the main or the accessory olfactory bulbs. As a result,
guidance cues responsible for the axonal projection pattern of
Grueneberg axons are still unknown.
Here, we took a genetic approach to first identify the
projection pattern of the Grueneberg ganglion, and found that
Grueneberg ganglion axons form homogeneously innervated
glomeruli among other necklace structures. It provided a unique
tool for identifying potential cues involved in Grueneberg axonal
wiring. We thus evaluated the role played by a guidance
molecule expressed by Grueneberg sensory neurons, neuropilin
1, in the elaboration of the Grueneberg projection map in the
olfactory bulb. We found that the expression of neuropilin 1 by
Grueneberg axons prevents them from innervating an area
restricted to the axonal projections of another chemosensor, the
vomeronasal organ.
MATERIALS AND METHODS
Animals
Mice were housed and handled in accordance with the guidelines and
regulations of the institution and of the state of Geneva. Nrp1flox/flox
animals were purchased from The Jackson Laboratory.
DEVELOPMENT
KEY WORDS: Olfactory system, Axon guidance, Neuropilin 1, Mouse, GC-G, GC-D
2784 RESEARCH ARTICLE
A zeocin-puromycin cassette flanked by flippase recognition target (FRT)
sites was inserted after an IRES-Cre-IRES-eGFP cassette. The resulting
construct was inserted by homologous recombination after the stop codon
of GC-G in BAC RP23-345B13 using methods described previously (Lee
et al., 2001). After the removal of the zeocin-puromycin cassette by
flippase in EL250 cells, the resulting bacterial artificial chromosome
(BAC) was used for pronuclear injections. Eight transgenic lines were
obtained, of which seven lines expressed the transgene in the Grueneberg
ganglion. Line 52, which exhibited robust GFP expression in the
Grueneberg ganglion, was used for further analysis.
Whole-mount analyses
After dissection of animals, whole-mount images of the Grueneberg
ganglion or the olfactory bulb were taken with a Zeiss SteREO Lumar
V12. z-stack images were projected to a single image by an extended focus
function of Axio Vision (Carl Zeiss).
Immunohistochemistry
Tissues were fixed with 4% paraformaldehyde (PFA) in PBS for 3 hours
(to 16 hours) at 4°C, followed by 25% sucrose in PBS overnight, and were
embedded in optimal cutting temperature compound (OCT). Cryostat
sections (10-20 m) were mounted on Superfrost + slides. Sections were
preincubated with PBS containing 0.3-0.5% Triton X-100 and 5% fetal calf
serum (FCS) for 30 minutes at room temperature, then incubated with
primary antibodies diluted in PBS with 0.3% Triton X-100 and 3% FCS
overnight at 4°C. The following primary antibodies were used; goat anticarbonic anhydrase II (CAII) (1:800, Santa Cruz Biotechnology), rabbit
anti-GFP (1:800, Abcam), chicken anti-GFP (1:800, Abcam), rabbit antiPDE2 (1:800, FabGennix), goat anti-OMP (1:400-800, Wako Laboratory
Chemicals), rabbit anti-V2R2 (1:200) (Martini et al., 2001), goat anti-Nrp1
(1:100-800, R&D Systems), rabbit anti-synaptotagmin (1:200, SigmaAldrich), rabbit anti- PGC-G (1:200, FabGennix), and goat anti-human
semaphorin 3A (1:20, R&D Systems). Slides were washed three times with
PBS and incubated with secondary antibodies for 2 hours at room
temperature. The following secondary antibodies were used; donkey Cy3conjugated anti-rabbit (1:800, Jackson Laboratory), donkey Alexa 555conjugated anti-goat (1:800, Invitrogen), chicken Alexa 488-conjugated
anti-rabbit (1:800, Invitrogen), goat FITC-conjugated anti-chicken (1:800,
Abcam), donkey Alexa 488-conjugated anti-goat (1:800, Invitrogen),
donkey Alexa 680-conjugated anti-rabbit (1:400, Invitrogen). Fluorescent
images were taken with a confocal microscope (Zeiss 510 Meta). All
sections were counterstained with DAPI (1 g/ml).
Whole-mount immunohistochemistry
Olfactory bulbs were fixed with 4% PFA in PBS for 2 hours and washed
with PBS for 20 minutes. Bulbs were dehydrated by graded
methanol/PBS series (25%, 50%, 100% methanol, 10 minutes for each
step). Bulbs were frozen at –80°C for 30 minutes and thawed at room
temperature for 10 minutes. This procedure was repeated three times.
Samples were rehydrated in 50%, 25% methanol/PBS series, washed
with PBS for 20 minutes and preincubated with PBS containing 0.5%
Triton X-100 and 5% FCS for 30 minutes at room temperature. This was
followed by an incubation with primary antibodies diluted in PBS with
0.5% Triton X-100 and 5% FCS for 72 hours at 4°C. Primary antibodies
were goat anti-carbonic anhydrase II (CAII) (1:1000, Santa Cruz
Biotechnology), rabbit anti-GFP (1:1000, Abcam). After washing with
PBS for 1 hour three times, bulbs were incubated overnight with
secondary antibodies donkey Alexa 555-conjugated anti-goat (1:1000,
Invitrogen) or chicken Alexa 488-conjugated anti-rabbit (1:1000,
Invitrogen). Fluorescent images were taken with a Zeiss SteREO Lumar.
V12. z-stack images were projected to a single image by an extended
focus function of Axio Vision (Carl Zeiss).
In situ hybridization
In situ hybridizations were performed as described previously (Roppolo et
al., 2007). Briefly, tissues were embedded in OCT and 14 m cryostat
sections were mounted on Superfrost Plus slides. Slides were fixed for 20
minutes with 4% PFA, and hybridized overnight at 65°C in the following
buffer: 1 ⫻ salt buffer [10 mM NaCl, 5 mM NaH2PO4, 5 mM Na2HPO4,
5 mM EDTA (pH 7.5)], 50% formamide, 10% dextran sulfate, 1 g/l
tRNA, 1 ⫻ Denhardt’s, with 20 ng/l cRNA probes. Following
hybridizations, slides were washed twice at 65°C and once at room
temperature with 1 ⫻ SSC, 50% formamide and 0.1% Tween20. Slides
were preincubated in 1 ⫻ MABT, 2% blocking reagent (Roche) for 20
minutes followed by 1 hour incubation with alkaline phosphatase-antidigoxigenin antibody (1:500, Roche) and peroxidase-anti-fluorescein
antibody (1:200, Roche). Peroxidase activity was detected by washing
slides three times with TNT [Tris 150 mM, NaCl 150 mM, Tween20
0.05% (pH 7.5)], incubation for 30 minutes with a biotinyl-tyramide
solution (PerkinElmer), three washes with TNT and incubation for 30
minutes with streptavidin-Alexa 488 (Invitrogen). Alkaline phosphatase
activity was detected by incubating slides with Fast Red substrate (DAKO)
for 30 minutes. Fluorescein- and digoxigenin-labeled RNA probes were
prepared using a DIG RNA Labeling Kit (Roche) following the
manufacturer’s instructions. The entire coding sequence of GFP was used
as probe. The Sema3a 3⬘UTR probe spanned nucleotides 122 to 1110
relative to the stop TGA. The sequences described in (Fleischer et al.,
2007) were used for TAAR6 and TAAR7 probes.
DiI labeling
DiI (7 l of a 250 g/ml solution, diluted in PBS from a 5 mg/ml DiI
solution dissolved in ethanol) was applied to one of the nostrils. DiI was
inhaled spontaneously. Thirty minutes later, the same amount of DiI
solution was applied to the other nostril. Animals were killed 2-3 days after
DiI administration, and the fluorescence of the caudal olfactory bulb was
analyzed by confocal microscopy (Zeiss 510 Meta).
RESULTS
A transgene that labels Grueneberg sensory
neurons
In order to visualize and discriminate the minute population of
Grueneberg ganglion neurons and their axonal projections in the
olfactory bulb, we took a transgenic approach. It involved the
insertion of a modified BAC into the mouse genome. This
transgene contained the guanylyl cyclase G gene, a gene that is
transcribed by Grueneberg neurons (Fleischer et al., 2009; Liu et
al., 2009; Chao et al., 2010) as early as postnatal day (P) 0
(supplementary material Fig. S1E). Among olfactory sensory
neurons, only those pertaining to the Grueneberg ganglion are
known to express this cyclase. A double polycistronic cassette
driving the expression of the GFP marker and of the Cre
recombinase was added to the cyclase gene (GCG-Cre-GFP
transgene; Fig. 1B). Eight GCG-Cre-GFP founders were
obtained. Seven GCG-Cre-GFP lines expressed the transgene at
different levels in the Grueneberg ganglion. A line with robust
expression in Grueneberg neurons was selected for further
analysis (Fig. 1C).
An exclusive labeling of V2R2-expressing neurons
The molecular receptors involved in the recognition of Grueneberg
stimuli are still elusive. However, a few potential chemoreceptors,
which include the vomeronasal panreceptor V2R2 (alternatively
known as V2R83), have been identified in Grueneberg neurons
(Fleischer et al., 2006b). V2R2 transcripts are found in virtually all
Grueneberg neurons in young mice and in adults. The GCG-CreGFP transgene labeled olfactory marker protein (OMP)
(supplementary material Fig. S1A-D) and V2R2-expressing
Grueneberg neurons in P7 mice (supplementary material Fig. S1FH). A few non-V2R2-expressing neurons were found to express
trace amine-associated receptors (TAARs) by in situ hybridization
(Fleischer et al., 2007). These were not labeled by the transgene
(supplementary material Fig. S1I,J). The number of these TAAR-
DEVELOPMENT
Generation of the BAC transgenic mice
Development 139 (15)
Fig. 1. Transgenic labeling of the Grueneberg ganglion neurons.
(A)Schematic of the olfactory system of a rodent. In blue, main
olfactory sensory neurons and axonal projections in the olfactory bulb.
In orange, vomeronasal organ and projections in the accessory olfactory
bulb. In green, Grueneberg ganglion and Grueneberg projections in the
necklace glomeruli. The medial part of the necklace is visible on the
figure. AOB, accessory olfactory bulb; GG, Grueneberg ganglion; MOE,
main olfactory epithelium; NG, necklace glomeruli; OB, olfactory bulb;
VNO, vomeronasal organ. (B)Schematic of the GCG-Cre-GFP
transgene. (C)Whole-mount ventral view of the nasal vestibule of a P6
mouse expressing the GCG-Cre-GFP transgene. Both Grueneberg
ganglion neurons and their axonal projections are GFP fluorescent. In
C, the blurry vertical structure that separates the right and left nasal
cavities corresponds to the nasal septum. Scale bars: 100m in C, left
panel; 20m in C, right panel.
positive cells was extremely low, and close to zero in adults. We
thus observed that for the transgenic line selected, the large
majority if not all of the Grueneberg sensory neurons were GFP
positive, whereas the fluorophore was absent from other olfactory
sensory populations (Fig. 2D; data not shown). This provided a
precise and unique tool to follow the Grueneberg axonal
projections, from the ganglion somata to the corresponding
glomeruli.
Homogeneous and early fasciculation of
Grueneberg axons
An indirect way to evaluate the molecular proximity between
olfactory subpopulations is to analyze their putative physical
association during axonal migration. Thus, in the vomeronasal
system, V1R- and V2R-expressing axons fasciculate together in
bundles while running along main olfactory axonal bundles, with
which they do not intermingle. This is also true between
subpopulations of main olfactory sensory neurons, which segregate
early during axonal fasciculation (Imai et al., 2009). We examined
RESEARCH ARTICLE 2785
Fig. 2. Axonal bundle formation of Grueneberg ganglion axons.
(A-C)Coronal section through the anterior part of the Grueneberg
projection fibers of a P7 GCG-Cre-GFP transgenic mouse. The fibers
(GFP positive) run bilaterally along the nasal septum and are grouped in
a single bundle (sometimes two). They do not appear to be
intermingled with non-OMP expressing axons. (D)Hemisection of a P7
GCG-Cre-GFP transgenic mouse head. (E-G)Coronal section of the
Grueneberg axonal bundle at the level of the main olfactory
neuroepithelium in a GCG-Cre-GFP mouse. The Grueneberg bundle
(GFP positive) runs deep under the main sensory epithelium, and is
associated with other, OMP-expressing (red) axonal bundles, but it does
not intermingle with them. Scale bars: 10m in C; 500m in D; 50m
in G.
Grueneberg fibers from GCG-Cre-GFP mice to evaluate their
possible joining and intermingling with other fibers. When leaving
the Grueneberg ‘grape-like’ ganglion, axons coalesced into a single
or a few bundles. At the tip of the nose, the bundle had a diameter
of about 20 m and was already dense, in the sense that it appeared
almost exclusively composed of Grueneberg fibers (Fig. 2A-C). It
then entered the area covered by the main olfactory epithelium,
where it ran under the neuroepithelium (Fig. 2D). It was found
associated with main olfactory bundles, but did not appear to
intermingle with main olfactory fibers (Fig. 2E-G).
Grueneberg ganglion glomeruli are present at
birth
The olfactory system is not mature at birth. This is true for both the
main olfactory and the vomeronasal systems. The sensory part of
the latter first contacts the outside world only a few days after birth.
To evaluate the developmental state of the Grueneberg system
neonatally, we looked for the potential presence of Grueneberg
ganglion axons and glomeruli in the olfactory bulb in neonates. We
analyzed six P0 GCG-Cre-GFP mice, and found, in all of them,
OMP-positive Grueneberg ganglion fibers that had coalesced in the
necklace glomeruli (Fig. 3A-D). These glomeruli were positive for
the presynaptic marker synaptotagmin (Fig. 3E), in contrast to most
olfactory glomeruli at this stage, suggesting an early maturation of
the Grueneberg circuitry. This early presence of Grueneberg
glomeruli was not detected in previous reports (Koos and Fraser,
2005), probably because of the use of less sensitive, non-genetic
DEVELOPMENT
Grueneberg ganglion axon wiring
2786 RESEARCH ARTICLE
Development 139 (15)
Fig. 3. Grueneberg ganglion glomeruli are present at birth.
(A)Dorsal view of an olfactory bulb from a P0 GCG-Cre-GFP transgenic
mouse. At birth, Grueneberg fibers are organized in a necklace shape
around the rostral part of the accessory olfactory bulb, and defined
GFP-expressing Grueneberg glomeruli (G) can already be observed.
(B-D)Sagittal section through a Grueneberg glomerulus from a P0
GCG-Cre-GFP transgenic mouse. (E)A Grueneberg glomerulus (G)
stains positively for Synaptotagmin (in red) on a sagittal section through
the olfactory bulb of a P0 GCG-Cre-GFP transgenic mouse. Scale bars:
200m in A; 50m in B,E.
approaches. Thus, unlike what is observed in other olfactory
sensory systems, the sensory part of the Grueneberg ganglion is
possibly functional at birth.
The topographical map of Grueneberg ganglion
glomeruli
We then aimed at a global analysis of the topographical map
formed by Grueneberg glomeruli in the olfactory bulb. Taking
advantage of the very superficial innervation of the bulb by
Grueneberg axons, we analyzed whole-mount preparations of
olfactory bulbs from GCG-Cre-GFP transgenic mice. We observed
dorsal, medial, ventral and lateral views of the bulbs of female P7
mice (n3) (Fig. 4A-D). After leaving the cribriform plate, the
Grueneberg bundle followed the medial side of the olfactory bulb
(Fig. 4B). Before reaching the necklace glomeruli, the bundle
divided in two (or sometimes more) smaller bundles, one aiming
at the dorsal part of the necklace, the other at the ventral part,
strictly avoiding the accessory olfactory bulb. A ventral view of the
bulb shows that fibers pertaining to the two bundles possibly rejoin
each other at the very bottom of the bulb (Fig. 4C). No stray fibers
were observed.
The lack of fixed structures for most views did not allow an
easy and precise comparison between individuals. We thus
decided to restrict our precise analysis of the topographical map
GC-G and GC-D axons project to different necklace
glomeruli
The necklace glomeruli are a composite group of neuropil-rich
structures. They are both innervated by Grueneberg ganglion axons
(which express the cyclase GC-G) and by main olfactory neurons
(which express the cyclase GC-D). These two sensory populations
not only share their peculiar necklace-shaped axonal projections
and the expression of a guanylyl cyclase, but they also express
elements of a cGMP second messenger pathway that includes the
cGMP-stimulated phosphodiesterase PDE2 and the nucleotidegated channel CNGA3 (Juilfs et al., 1997; Meyer et al., 2000;
Mamasuew et al., 2008; Fleischer et al., 2009; Liu et al., 2009).
This naturally suggests potential parallels in terms of signal
processing or function between these sensory populations. Owing
to these intriguing common characteristics, and to the coconvergence of similar but not identical olfactory fibers in specific
necklace glomeruli (Cockerham et al., 2009), we investigated the
potential crosstalk (i.e. co-convergence) between GC-D and
Grueneberg axonal projections (Fig. 5A). In order to discriminate
between these two types of glomeruli in the necklace structure, we
took advantage of a peculiar characteristic of Grueneberg neurons:
in live mice, Grueneberg sensory neurons do not incorporate DiI
when the dye is introduced into the nostrils, possibly owing to their
lack of direct contact with the outside world. By contrast, main
olfactory sensory neurons, including GC-D-positive cells,
incorporate the dye, which is readily transported toward the bulb.
We took this approach to label olfactory sensory neurons from P5P7 transgenic mice (n9). We thus obtained mice with green and
red fluorescent Grueneberg ganglion and GC-D/main olfactory
neuron axonal fibers, respectively. No co-innervation between the
two populations was observed (Fig. 5B,C). We further confirmed
the separate nature of the Grueneberg and main olfactory glomeruli
by immunohistochemistry with the specific GC-D and necklace
markers carbonic anhydrase II (CAII) and PDE2, respectively (Fig.
5D,E). Thus, Grueneberg ganglion neurons and GC-D sensory
neurons, although both targeting the necklace glomeruli, do not
wire, nor converge together, suggesting a parallel processing of
information.
DEVELOPMENT
to the glomeruli that were visible from the dorsal view of the
bulb, a view that benefited from the fixed position of the
accessory olfactory bulb. We compiled the dorsal glomerular
pattern of six animals (supplementary material Fig. S2A).
Grueneberg glomeruli were disposed along the entire length of
the necklace glomeruli, and did not exhibit any specific
organization, or at least no zones in which glomeruli would be
more likely to form. We then evaluated a possible evolution of
the pattern with age, and analyzed mice at weaning (P21), and
adult animals (Fig. 4E-G). We drew maps similar to the ones
generated for P7 mice (supplementary material Fig. S2B-D), and
observed, again, no specific zone of convergence. The number
of glomeruli was stable from one week to adulthood: at P7
(9.9±1.6), at P21 (9.7±1.8) and at P60 (10.8±1.5) (Fig. 4H). We
did not observe differences in number of glomeruli or projection
patterns between genders (supplementary material Fig. S2C,D).
These data show that Grueneberg axons project around the entire
olfactory trunk, unlike previous reports that only found
innervation of the dorsal part of the necklace by Grueneberg
fibers (Fuss et al., 2005; Roppolo et al., 2006). Again, similar to
our identification of Grueneberg glomeruli in newborns (which
was not observed by others), it probably reflects poorly sensitive
tracing methods that were used in the past.
Grueneberg ganglion axon wiring
RESEARCH ARTICLE 2787
Grueneberg ganglion axons project dorsally
relative to GC-D axons
The narrow band occupied by the necklace glomeruli appears to be
composed of glomeruli randomly organized along its width. We
took a close look at this question and examined dorsal views of the
olfactory bulb. We recorded the position of Grueneberg ganglion
and GC-D glomeruli in seven bulbs of 3-week-old animals. Both
Grueneberg ganglion axons and glomeruli were found rostralized
relative to those of GC-D-expressing neurons on the dorsomedial
part of the necklace (Fig. 6A-D). This topographic exclusion was
not observed or was unclear in other parts of the necklace
(supplementary material Fig. S3A-C). This observation suggests
that the guidance cues that direct the projection map of Grueneberg
ganglion and GC-D axons are not identical.
Neuropilin 1-dependent wiring of Grueneberg
ganglion axons
In order to investigate the factors involved in the peculiar
organization of Grueneberg ganglion axons, we evaluated
Grueneberg sensory neurons for potential expression of neuropilin 1,
a guidance molecule that was shown to be involved in the wiring of
specific olfactory populations, in particular in pre-target axon sorting
(Imai et al., 2009). We found expression of neuropilin 1 in
Grueneberg axons and glomeruli in neonates (Fig. 7A,B). In
Grueneberg glomeruli of older animals (3 weeks old), the presence
of the protein was no more, or barely detectable in Grueneberg
glomeruli (data not shown). We did not detect neuropilin 1
expression in the accessory olfactory bulb (Fig. 7B; data not shown).
In the main olfactory system, the level of neuropilin 1 expression is
heterogeneous and is dependent on the odorant receptor expressed
by each sensory neuron. This heterogeneous expression is crucial in
the axonal guidance process of these sensory neurons. We assessed
whether a similar heterogeneous neuropilin 1 expression was present
in Grueneberg axonal fibers, and found homogeneous levels of
expression between Grueneberg axons (data not shown).
We then explored the expression pattern of the main neuropilin
1 ligand, Sema3a, in the vicinity of the target of Grueneberg axonal
projections, i.e. around the necklace glomeruli. We made two
observations at the time Grueneberg glomeruli are formed, between
embryonic day (E) 19 and P5. First, mitral cells that innervate
glomeruli of the main olfactory and accessory olfactory bulbs
transcribe Sema3a (Fig. 7C-E). These transcripts were not present
in cells located directly beneath Grueneberg glomeruli (Fig. 7C-E),
thus creating a narrow semicircular band devoid of Sema3a
transcripts that correlates with the position of the rostral necklace
glomeruli. Second, vomeronasal sensory neurons transcribe
Sema3a at P0 (Fig. 7F), a transcription reflected by its
corresponding protein in vomeronasal axonal fibers in the
accessory olfactory bulb (Fig. 7G). The distribution of the Sema3a
protein in the AOB is thus broader than the in situ data suggest.
To evaluate the potential role played by neuropilin 1 in
Grueneberg axonal wiring, we first generated a mouse bearing a
neuropilin 1 null allele (Nrp1del), by crossing a line bearing a
neuropilin floxed allele (Nrp1flox) to a strain expressing the Cre
recombinase constitutively. We then took advantage of the GCGCre-GFP transgenic line we produced, which in addition to the
GFP fluorophore also drives the expression of the Cre
recombinase. We generated Nrp1del/flox;GCG-Cre-GFP and
Nrp1flox/flox;GCG-Cre-GFP animals, which were missing
neuropilin 1 exclusively in Grueneberg ganglion neurons. The
reason for using compound Nrp1 heterozygotes was to facilitate
the Cre-mediated generation of two null alleles. We first
evaluated potential defects in axonal bundle formation of
Nrp1del/flox;GCG-Cre-GFP and Nrp1flox/flox;GCG-Cre-GFP mice.
We found no alterations in their compactness or potential
intermingling with non-like fibers (supplementary material Fig.
S4D,E). We then analyzed, in whole mounts, the corresponding
Grueneberg axonal projections. As a control, we evaluated the
projections of Nrp1+/del;GCG-Cre-GFP and of Nrp1+/flox;GCGCre-GFP mice. We could not distinguish them from those of
Nrp1+/+;GCG-Cre-GFP mice (Fig. 8A,B; data not shown). By
contrast, all Nrp1del/flox;GCG-Cre-GFP animals analyzed
exhibited a striking alteration of the Grueneberg projections
(n6). The formation of Grueneberg glomeruli and their
exclusion from GC-G glomeruli was not affected (supplementary
material Fig. S4A-C), but a large number of Grueneberg axonal
DEVELOPMENT
Fig. 4. Topographical distribution of Grueneberg
ganglion glomeruli. (A-D)Whole-mount, dorsal,
medial, ventral and lateral views of the Grueneberg
ganglion glomeruli in a P7 GCG-Cre-GFP transgenic
mouse. The endogenous GFP fluorescence from the
GCG-Cre-GFP transgene is readily visible. The
Grueneberg axonal projection bundle separates into
two before reaching the accessory olfactory bulb. The
Grueneberg fibers and glomeruli then circle the entire
trunk of the olfactory bulb, and the fibers rejoin at the
base of the olfactory trunk. (E-G)Dorsal views of
Grueneberg glomeruli from P21 and P60 male and
female GCG-Cre-GFP transgenic mice. (H)Number of
Grueneberg glomeruli in P7, P21 and adult GCG-CreGFP transgenic mice. Scale bars: 500m in A-G.
2788 RESEARCH ARTICLE
Development 139 (15)
Our results thus support a key role played by neuropilin 1, in the
avoidance of Grueneberg neurons of the accessory olfactory bulb.
Fig. 5. Main olfactory and Grueneberg neurons project to
different necklace glomeruli. (A)Two models of olfactory integration
in the necklace glomeruli. One involves exclusively parallel lines of
coding between GC-D and GC-G-expressing neurons, the other signal
integration at the level of the glomeruli. (B,C)Whole-mount, dorsal
view of the necklace glomeruli of a P7 transgenic mouse. Grueneberg
glomeruli are in green (GFP fluorescent) and axonal projections from
the main olfactory epithelium are labeled in red (DiI labeled).
(D)Sagittal section through the necklace glomeruli of a 3-week-old
GCG-Cre-GFP transgenic mouse. Grueneberg glomeruli are labeled in
green and GC-D glomeruli (CAII positive) are in red. (E)Sagittal section
through the necklace glomeruli of a 3-week-old GCG-Cre-GFP
transgenic mouse. Both Grueneberg and GC-D glomeruli express
PDE2A. Scale bars: 200m in B,C; 50m in D,E.
bundles, composed of multiple axons, did not avoid the
accessory olfactory bulb as they do in wild-type animals: they
crossed it from part to part (Fig. 8C-F). Some Grueneberg fibers
entered deep into the accessory olfactory bulb glomerular layer
(Fig. 8E). This lack of exclusion by vomeronasal projections was
mostly observed on the rostral part of the accessory olfactory
bulb. As the absence of neuropilin 1 was restricted to
Grueneberg ganglion neurons, the observed phenotype did not
result indirectly from the alteration of other cell types lacking
neuropilin 1.
A second type of neuropilin, neuropilin 2, has been shown to
play a crucial role in the guidance of axons from olfactory sensory
neurons present in the main olfactory epithelium, and in particular
GC-D-expressing axons (Walz et al., 2007). To evaluate the
potential role of this neuropilin in guiding Grueneberg axons, we
took a genetic approach, similar to the one employed for the
specific deletion of neuropilin 1 in GC-G-expressing neurons. We
found that Grueneberg glomeruli were well formed in these
mutants, and no Grueneberg fibers crossing the accessory olfactory
bulb were observed (n8) (supplementary material Fig. S4F).
A specific circuitry for Grueneberg axons
We first took a look at the way Grueneberg ganglion axonal
projections leave the anterior nasal vestibule. We observed an early
fasciculation of Grueneberg fibers into one or two axonal bundles.
This fasciculation was not affected by the later joining of axonal
bundles from the main olfactory epithelium, and remained
homogeneous until the fibers reached the caudal part of the
olfactory bulb. This early segregation between groups of axonal
fibers before reaching their target is consistent with reports that
show in the main olfactory system an early sorting of axons
pertaining to different olfactory sensory neuron subpopulations
(Imai et al., 2009). Grueneberg axons are thus likely to be
recognized by main olfactory bundles as a non-like group of
olfactory fibers. After crossing the cribriform plate and running
along the medial olfactory bulb, Grueneberg axons project to a
unique chain of glomeruli, the necklace glomeruli, and surround
the entire trunk of the olfactory bulb. The peculiar morphological
characteristics of the Grueneberg ganglion sensors are thus
reflected at the level of the Grueneberg circuitry, at least up to the
olfactory bulb.
The role played by the Grueneberg system is still unclear and
is apparently multimodal. A few observations may provide
insights into this question. The first observation is linked with
the maturation of the Grueneberg system, which we found
unusual, as Grueneberg synapse-containing glomeruli are already
present in newborn animals. This early maturation of axonal
wiring is not the rule for olfactory sensors. Many glomeruli from
the main olfactory system are indeed formed during and after the
perinatal period. Vomeronasal glomeruli also appear late,
probably reflecting the postnatal maturation of this system (in
mice, the duct that connects the vomeronasal organ with the
outside world opens after the first week of life). The apparent
maturity of the Grueneberg system at birth is thus potentially
functionally meaningful. It would be consistent with the known
activators of the Grueneberg ganglion: namely ‘fear’-induced
compounds and cold temperature, two stimuli that could trigger
stress, and perception of which could be beneficial to animals of
all ages, including newborns. Possibly linked to the latter
stimulus, cold temperatures, which induce c-fos transcription in
the Grueneberg ganglion of neonates (Mamasuew et al., 2008),
trigger ultrasonic vocalizations that are attractive stimuli to the
lactating mother. A second observation that may provide insight
into the role played by the Grueneberg system may reside in its
wiring pattern. The innervation of the bulb by Grueneberg fibers
is reminiscent of olfactory subsystems processing socially
relevant chemical information. We found about ten Grueneberg
DEVELOPMENT
DISCUSSION
By combining a transgenic approach with imaging techniques, we
performed an analysis of the axonal wiring of the Grueneberg
ganglion, one of the rodent olfactory subsystems. We show that the
necklace glomeruli are composed of homogeneous units,
innervated by either one of two peculiar sensory populations: the
GC-D-expressing neurons from the main olfactory system and
those from Grueneberg ganglion. Further, taking advantage of our
transgenic tool associated with conditional null alleles, we then
show that the expression by Grueneberg sensory neurons of a
specific guidance molecule, neuropilin 1, is involved in the
establishment of this wiring diagram.
Grueneberg ganglion axon wiring
RESEARCH ARTICLE 2789
necklace glomeruli per bulb, apparently connected together. This
type of wiring is unlike the canonical projection pattern observed
in the main olfactory system, but is reminiscent of the one found
in the vomeronasal system, where the numerous like glomeruli
are often linked together, and is very similar to the one
corresponding to GC-D-expressing neurons. Knowing that both
vomeronasal and GC-D neurons are specialized in interpreting
innate and/or socially relevant signals (Halpern and MartinezMarcos, 2003; Munger et al., 2010), it naturally suggests that
Grueneberg neurons play a role in inter-individual interactions.
Making parallels between these different systems should,
however, be tempered. Necklace glomeruli are unusual in their
innervation: they receive heterogeneous sensory input from main
olfactory neurons. They integrate axonal projections from GC-Dexpressing neurons, and from another, still elusive population
expressing OMP (Cockerham et al., 2009). This very unusual
stimulus coding strategy (that is for the sensory part of the olfactory
system), which involves multiple and functionally distinct sensory
neuron subtypes, led to the suggestion that Grueneberg glomeruli
could also share this heterogeneous innervation pattern. We report
here homogeneously innervated Grueneberg glomeruli. There is
thus no direct crosstalk between Grueneberg axonal projections and
other types of olfactory sensory projections, at least until the first
projection relay. Thus, the Grueneberg system, although probably
involved in innate and/or social interactions like GC-D neurons,
possibly plays a different role.
Neuropilin 1 and Grueneberg axonal projections
How the olfactory maps in the bulb are built is still unclear. The
adequate targeting of a functionally homogeneous olfactory
sensory axonal population (i.e. expressing a given olfactory
chemoreceptor) first requires a capacity to reach a defined area in
the olfactory bulb, followed by an ability to coalesce. A few
molecules have been shown to play crucial roles in both
glomerulus formation, and in the topographical position of these
glomeruli (Cho et al., 2007; Sakano, 2010). In the main olfactory
system, in a given sensory neuron, expression of several of these
molecules is dependent on the olfactory receptor gene transcribed.
It was proposed that each olfactory receptor defines a specific level
of cAMP in the olfactory sensory neuron, which in turn drives the
expression of a specific set of homophilic adhesion/repulsion and
axon guidance molecules, such as kirrels, ephrins, and neuropilin
1 (Serizawa et al., 2006; Cho et al., 2007; Imai et al., 2006; Imai et
al., 2009; Sakano, 2010; Takeuchi et al., 2010). These guidance
receptors can exert a direct effect on the sensory neurons that
express them, or can affect the targeting of other neurons.
Most of the studies aimed at understanding olfactory guidance
mechanisms targeted the main and the vomeronasal olfactory
systems. Thus, the axonal guidance signals that are involved in the
establishment of the narrow chain of necklace glomeruli are almost
entirely unknown today.
As for the rest of the olfactory map, the formation of the
necklace glomeruli surely relies on distinct sets of guidance cues,
involved in their general targeting and their ability to coalesce.
The very peculiar trajectory of necklace glomeruli around the
olfactory bulb suggests either a strong attractive signal present
on this path, and/or concomitant repulsive signals from the
structures flanking the glomerular chain (i.e. from the main
olfactory and the accessory olfactory bulbs, respectively) to
maintain it sandwiched in between. A report suggests that the
repulsive hypothesis is correct, at least for the avoidance of
structures located rostrally relative to the necklace: a deficiency
in expression of neuropilin 2 alters the projection map formed
by GC-D-expressing neurons in the necklace glomeruli in mice
(Walz et al., 2007). In these null animals, some of the GC-D
axonal projections miswire and end their course earlier, in the
glomerular layer of the main olfactory bulb. Neuropilin 2 plays
thus a role in preventing GC-D axons from arborizing too early,
and in innervating rostral areas of the main olfactory bulb. Our
observations point to a signal that plays a reverse but
complementary role, that is to prevent the overshooting of the
Grueneberg axonal projections past the necklace. We found that
at a time Grueneberg sensory axons navigate toward the bulb,
they express the neuropilin 1 protein. The genetic deletion of its
corresponding gene, when restricted to Grueneberg neurons,
allows Grueneberg axons to cross the area occupied by the
accessory olfactory bulb, an area that is usually entirely devoid
of Grueneberg fibers. The overshooting control is thus at least
partly played by neuropilin 1. That is the first step in axonal
guidance, the general positioning, and not the second step that
affects coalescence, which is apparently independent of this
protein (as we did not observe co-convergence of Grueneberg
axons lacking neuropilin 1 and GC-G axons).
It is tempting to speculate that our miswiring observation results
from the lack of response by Grueneberg axons to a repulsive
neuropilin 1 agonist such as Sema3a in the accessory olfactory
bulb. We in fact observed an expression pattern of this repulsive
molecule in mitral and sensory vomeronasal cells that would be
consistent with this hypothesis.
Our finding thus sheds light into one of the two guidance
processes that direct the establishment of the olfactory maps: the
one that is responsible for the restriction of axonal fibers at a
DEVELOPMENT
Fig. 6. Dorsomedial Grueneberg ganglion axons
project rostrally relative to GC-D axons.
(A-C)Topographical position of Grueneberg (green) and
GC-D (red) glomeruli on a dorsal view of the necklace
glomeruli from the left bulb of 3-week-old GCG-CreGFP transgenic mice. GC-D fibers were immunolabeled
in red with an anti-CAII antibody and Grueneberg axons
were immunolabeled in green with an anti-GFP
antibody. (D)The glomerular position of Grueneberg
(green) and GC-D (red) glomeruli corresponding to a
dorsal view of the necklace glomeruli of seven bulbs
from GCG-Cre-GFP mice is shown. Scale bar: 500m
in A.
Development 139 (15)
Fig. 7. Neuropilin1 and Sema3a expression in Grueneberg axons
and the vomeronasal system. (A)Grueneberg axons (GFP positive)
express neuropilin 1 (in red) at P0. (B)Grueneberg glomeruli (G, red and
green and thus yellow) and glomeruli from the MOE (red) are positive
for neuropilin1, unlike glomeruli and mitral cells from the AOB. (C-E)In
situ hybridizations on sagittal sections of olfactory bulbs, showing
Sema3a transcripts in mitral cells innervating main and accessory
olfactory bulb glomeruli, but not in mitral cells innervating necklace
glomeruli. E19 (C), P0 (D) and P5 (E) animals. No signal was observed in
vomeronasal MT cells with a sense Sema3a probe (inset in D) or
antisense GFP or OMP probes (not shown). A Grueneberg glomerulus
(G, in green, in a GCG-Cre-GFP transgenic mouse) is visible in E. (F)In
situ hybridization showing Sema3a transcripts in vomeronasal neurons
in a P0 mouse. (G)The entire glomerular layer of the accessory olfactory
bulb is Sema3a-immunopositive at P0. AOB, accessory olfactory bulb;
Ga, Grueneberg axonal tract; L, lumen; G, Grueneberg glomerulus;
MOE, main olfactory epithelium; MT, mitral cell; VNO, vomeronasal
organ. Scale bars: 50m in A-E.
Fig. 8. The wiring of Grueneberg fibers is dependent on
neuropilin 1. (A)Dorsal view of the Grueneberg glomeruli from the
left bulb of a Nrp1+/+;GCG-Cre-GFP 3-week-old mouse. (B)Dorsal view
of the Grueneberg glomeruli from the left bulb of a Nrp1+/del;GCG-CreGFP 3-week-old mouse. (C,D)Dorsal view of the Grueneberg glomeruli
from the left bulb of two Nrp1flox/del;GCG-Cre-GFP 3-week-old mice.
Grueneberg axonal fibers (arrowheads) and microglomeruli (asterisks)
that cross the rostral part of the accessory olfactory bulb are visible.
(E)Section through the accessory olfactory bulb of a 3-week-old
Nrp1flox/del;GCG-Cre-GFP mouse. A Grueneberg glomerulus (G) is
visible, together with two Grueneberg fibers that end deep into the
vomeronasal glomeruli (white arrows). (F)Number of Grueneberg
axonal bundles (each containing multiple axons) crossing the AOB from
the medial to the lateral side in Nrp1+/+;GCG-Cre-GFP and
Nrp1flox/del;GCG-Cre-GFP mice. *P<0.0001. AOB, accessory olfactory
bulb; G, Grueneberg glomerulus. Scale bars: 200m in A-D; 100m
in E.
specific place. What remains to be found (something that is also
lacking for the other olfactory subsystems), is a comprehensive
understanding of all players involved. These are undoubtedly
specific although partially overlapping for each olfactory
subpopulation, as their expression leads to highly stereotyped and
specific projection patterns.
DEVELOPMENT
2790 RESEARCH ARTICLE
Acknowledgements
The authors thank Roberto Tirindelli for the generous gift of the anti-V2R2
antibody. We also acknowledge members of the laboratory for discussions and
reagents.
Funding
T.M. was supported by a Japan Society for the Promotion of Science (JSPS)
postdoctoral fellowship. This work was funded by the University of Geneva,
the Swiss National Research Fund [grants 310030E-135910 and 31003A132919] and the National Research Center Frontiers in Genetics.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.077008/-/DC1
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Grueneberg ganglion axon wiring