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First published online 30 May 2007
doi: 10.1242/dev.006346
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1 Department of Zoology and Animal Biology, and NCCR Frontiers in Genetics,
University of Geneva, Geneva, Switzerland.
2 Department of Pharmacology, University of Washington, Seattle, WA 98195-7280,
USA.
Author for correspondence (e-mail:
Ivan.Rodriguez{at}zoo.unige.ch)
Accepted 13 April 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Olfaction, Axon guidance, Adenylyl cyclase 3, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The first step in olfactory processing in the main olfactory system
involves the recognition of chemical stimuli by odorant receptors. These are
seven transmembrane proteins expressed on dendritic endings of olfactory
sensory neurons (OSNs) (Buck and Axel,
1991
). Each OSN expresses a single or a few odorant receptor genes
among a repertoire of over a thousand in the mouse
(Chess et al., 1994). Odorant
receptor activation leads to a transduction cascade involving the G protein
alpha subunit G
olf, followed by the activation of adenylyl cyclase 3
(adenylate cyclase 3; Ac3) and the cAMP-dependent opening of an heteromeric
cation permeable channel containing the Cnga2 subunit
(Ronnett and Moon, 2002).
Calcium entry into the OSN then activates a chloride current that helps to
depolarize the neuron.
OSNs project their axons towards spherical neuropil-rich structures called
glomeruli, located in the cortical layer of the olfactory bulb
(Mombaerts et al., 1996;
Vassar et al., 1994). To each
functionally identical OSN population (i.e. expressing the same odorant
receptor) correspond only two glomeruli per bulb. The topographical position
of these two structures is conserved between the right and left bulbs, and
between different individuals. Thus, the mouse olfactory bulb is composed of
thousands of glomeruli, organized in a well-defined and relatively fixed
topographical map.
Unlike that observed for most mammalian neuronal structures, the wiring process leading to the establishment of this remarkable bulbar map is not limited to a narrow embryonic phase. Targeting of OSNs towards the bulb is indeed a lifelong continuous process, which compensates for the unusually (for neurons) short lifespan of OSNs.
How is this precise map elaborated and maintained? This process is not yet
understood, but mounting evidence points to activity-dependent mechanisms
(Yu et al., 2004;
Zou et al., 2004). Odorant
receptors (Mombaerts et al.,
1996; Wang et al.,
1998), G protein subunits
(Imai et al., 2006) and Pka
(Yoshida et al., 2002) have
been reported to play a role in OSN targeting to the olfactory bulb. Two
recent reports propose that odorant receptor-mediated signals direct axonal
targeting of OSNs via the activation of its associated G protein and the
production of cAMP (Chesler et al.,
2007; Imai et al.,
2006). However, analyses of mouse mutants lacking key elements of
the odorant transduction cascade such as Golf or Cnga2 have failed to
reveal major targeting defects of OSNs
(Belluscio et al., 1998;
Zhao and Reed, 2001;
Zheng et al., 2000); except
for Ac3, which has been linked to a disorganization of the glomerular layer in
the bulb (Chesler et al., 2007;
Trinh and Storm, 2003).
We report here that Ac3 is a likely key element in the odorant-receptor-mediated axonal guidance, as its lack drastically affects the reading of the olfactory bulb coordinates by OSN axonal projections, and prevents the expression of a major axon olfactory axon guidance molecule, neuropilin 1.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Mombaerts et al., 1996;
Potter et al., 2001;
Vassalli et al., 2002;
Wong et al., 2000;
Rodriguez et al., 1999). Mice
were bred on a C57BL6 background and animals analyzed were issue from
Ac3+/- crosses. Age- and sex-matched littermates were used when
comparing mutant and wild-type phenotypes. Animals were housed and handled in
accordance with the guidelines and regulations of the institution and of the
state of Geneva.
In situ hybridizations
Tissues were embedded in OCT without fixation, and 14 µm cryostat slices
were mounted on Superfrost+ slides (Menzel-Glaser), dried for 40 minutes,
fixed for 20 minutes at 4°C with 4% paraformaldehyde and hybridized
overnight at 65°C in the following buffer: 1 x salt buffer (10 mM
NaCl, 5 mM NaH2PO4·H2O, 5 mM
Na2HPO4·2H2O, 5 mM EDTA, pH 7.5), 50%
formamide, 10% dextran sulphate, 1 µg/µl tRNA, 1x Denhardt's, with
20 ng/µl cRNA probe(s). The Ac3 probe contained a 604 bp fragment starting
60 bp before the start codon. For M72 (Olfr160;
M72-lacZ) and P2 (Olfr17;
P2-lacZ), or MOR23 (Olfr16;
MOR23-GFP) and Omp (Omp-GFP)
transcript identification, probes containing a 1.8 kb HindIII
fragment of lacZ or the entire GFP ORF were used, respectively.
Following hybridizations, slides were washed twice at 65°C and once at RT
with 1x SSC, 50% formamide and 0.1% Tween 20. Slides were pre-incubated
in 1x MABT, 2% blocking reagent (Roche) for 30 minutes followed by a 1
hour incubation with alkaline phosphatase-anti-digoxigenin antibody (1:1000,
Roche) or/and peroxidase-anti-fluorescein antibody (1:200, Roche). Peroxidase
activity was revealed by washing slides three times with TNT (150 mM Tris, 150
mM NaCl, 0.05% Tween 20, pH 7.5), incubation for 30 minutes with a
biotinyl-tyramide solution (PerkinElmer, 1:50), three washes with TNT and
incubation for 30 minutes with streptavidin-Alexa 488 (Invitrogen, 1:100).
Alkaline phosphatase activity was detected by incubating slides with Fast Red
substrate (DAKO) for 30 minutes. Sections were mounted in DABCO mounting
medium (Sigma). Fluorescein and digoxigenin-labeled RNA probes were prepared
using the DIG RNA Labeling Kit (Roche) following the manufacturer's
instructions.
Microscopy
Images were taken on a Zeiss Axioplan2 wide field microscope or on a Leica
SP2 confocal microscope. All sections were counterstained with DAPI (5
minutes, 1 µg/ml) and slides were mounted using DABCO anti-fade mounting
medium.
Whole-mount analyses
Animals were dissected, the heads fixed by immersion in 4% paraformaldehyde
at 4°C for 20 minutes and stained with X-Gal as previously described
(Rodriguez et al., 1999).
Whole-mount images were taken on a Leica MZFLIII or Zeiss SteREO Lumar.V12
fluorescence-equipped binoculars.
RT-PCRs
Tissues were harvested and immediately processed using the Qiagen RNeasy
Mini Kit. The following primers were used to amplify Ac3 transcripts:
5'-ATCCCAAATTCCGGGTCATCAC-3' and
5'-GGAAGCCGTACTCTCGAAGGAT-3'. Amplifications were performed under
the following conditions: 5 minutes at 95°C, followed by 34 cycles of 1
minute at 95°C, 1 minute at 60°C, and 2 minutes at 72°C, and a
final extension of 10 minutes at 72°C.
Western blots
Tissues were homogenized in 10 mM Tris-HCl (pH 7.6), 1 mM EDTA, 100 mM
NaCl, 100 µg/ml PMSF, 1 µg/ml aprotinin. Protein extracts (15 µg)
were loaded on 10% Bis-Tris NuPAGE gels (Invitrogen), and transferred to PVDF
membranes. Membranes were incubated for 16 hours with an anti-Ac3 antibody
(1:500, sc-588 Santa Cruz Biotechnology), followed by incubation with an
alkaline phosphatase-conjugated anti-rabbit antibody. The phosphatase was
revealed with the CDP-Star chemiluminescent substrate (WesternBreeze,
Invitrogen).
Immunohistochemistry
Tissues were fixed for 2 hours (Ac3) or overnight (neuropilin 1, NCAM) in
4% paraformaldehyde and embedded in OCT. Cryostat slices (10-14 µm) were
mounted on Superfrost+ slides. Slides were pre-incubated in 0.5% Triton, 10%
FCS (except for anti-Ac3: 0.1% FCS), 1x PBS for 30 minutes at room
temperature. Goat anti-rodent-Omp (1:200-800, Wako Laboratory Chemicals),
rabbit anti-mouse-Ac3 (1:400, sc-588 Santa Cruz Biotechnology), mouse anti-rat
NCAM (1:500, Sigma) and goat anti-rat-Neuropilin (1:100, AF566, R&D
Systems) antibodies were used. After overnight incubation at 4°C, slides
were washed three times with 0.5% Triton, 1x PBS for 10 minutes and
stained with DAPI for 5 minutes (1 µg/ml). Primary antibodies were revealed
with Alexa 488-conjugated donkey anti-goat (1:200-800, Invitrogen),
Cy3-conjugated donkey anti-rabbit (1:250, Jackson Laboratories), Alexa
555-conjugated donkey anti-mouse (1:500, Invitrogen) or Alexa 555-conjugated
donkey anti-goat (1:800, Invitrogen) secondary antibodies. Slides were mounted
with antifade reagent (DABCO). To evaluate the specificity of the anti-Ac3
antibody, the peptide used to raise the antibody (Santa Cruz) was
pre-incubated for 2 hours at room temperature with the Ac3 antibody [10x
excess (w/w)].
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Wong et al.,
2000). We found it present from embryogenesis to adulthood in
OSNs, both at the mRNA (Fig.
1A,C), and protein levels (Fig.
1B,E-H). As expected, the protein was highly enriched in OSN
dendritic endings (Fig. 1H),
but was barely detectable in OSN somata. Surprisingly, immunohistochemical
analysis of axonal projections comprising the nerve layer and glomeruli
indicated that Ac3 was present in these structures
(Fig. 1H), although at a lower
concentration than the one observed in dendritic endings. Transcription of Ac3
by OSNs was observed from the early stages of OSN axonal pathfinding
(Fig. 1A). The corresponding
protein was also identified early during development, at embryonic day 12.5
(E12.5) in OSN dendrites and at E16 in the nerve layer
(Fig. 1E).
Western blot analysis of Ac3 OSN and olfactory bulb extracts showed the
absence of the major olfactory glycosylated Ac3 form in the bulb (200-210 kD)
and the presence of another Ac3 variant, of lower apparent molecular weight
(155-170 kD) (Fig. 1B). A
short, possibly similar, Ac3 variant has previously been reported in male germ
and smooth muscle cells (Gautier-Courteille
et al., 1998; Wong et al.,
2001).
We then investigated the possible Ac3 expression in sensory neurons pertaining to the vomeronasal system, a system that theoretically does not transduce chemosensory signals via cAMP. We found a weak expression of the Ac3 transcript in the vomeronasal neuroepithelium (Fig. 1D).
Ac3-deficient OSNs target aberrantly to the olfactory bulb
As previously mentioned, mice bearing null alleles of genes coding for the
major elements of the odorant signal transduction cascade (Golf)
exhibit, if any, only weak alterations of the topographical map in the
olfactory bulb (Belluscio et al.,
1998; Zhao and Reed,
2001; Zheng et al.,
2000). These observations, supporting the argument against a role
played by part of the odorant-induced cascade in guidance, possibly reflect a
partial knockout rescue by molecules still present in OSNs, such as the
ubiquitously expressed Gs. To test this caveat, we investigated the
potential axon guidance alterations resulting from the lack of Ac3 in
OSNs.
To identify a potential global effect of the cyclase on the whole olfactory
sensory population, we first visualized the entire OSN population and its
axonal projections, by crossing a line deficient in Ac3
(Wong et al., 2000) and a
transgenic mouse line (Omp-lacZ) in which all mature OSNs
express ß-galactosidase (Mombaerts et
al., 1996). Whole-mount analyses of Ac3-/- olfactory
bulbs showed a strikingly altered organization of glomeruli in the bulbs. In
control animals, glomeruli cover the entire olfactory bulb and are often
overlapping, rendering their individual identification in whole mounts
difficult (Fig. 2A,C,D). The
dorsocaudal part of the olfactory bulbs from P15 Ac3-/- animals
were almost devoid of Omp-positive glomeruli
(Fig. 2B,E,F), revealing some
rare caudally located glomeruli apparently unaffected and easily identifiable
because of the absence of surrounding Omp-expressing glomeruli. Specific
glomeruli, known as the necklace and Grueneberg glomeruli, which are located
at the very caudal part of the olfactory bulb, appeared loosely organized in
the mutants (Fig. 2E,F).
Sagittal sections through olfactory bulbs confirmed that not only the global
glomerular map was affected in Ac3 mutants, but also, at the glomerular level,
the individual structure of glomeruli, which appeared disorganized
(Fig. 2G,H).
|
). Did the
glomerular map alteration observed in Ac3-/- animals reflect the
absence of the OSN population projecting to the caudal part of the bulb? We
analyzed by whole mounts the olfactory sensory area in Omp-lacZ
Ac3-/- mice to identify potential zones devoid of
Omp-expressing neurons, but did not identify any specific epithelium area
lacking mature OSNs (Fig.
2A,B).
The weak Ac3 expression in the vomeronasal organ then led us to investigate
a potential role, possibly similar to the one we observed in the main
olfactory system, also played by this protein in the wiring of vomeronasal
sensory neurons. We analyzed whole mounts of Ac3-/-
Omp-lacZ accessory olfactory bulbs (10 bulbs from P10-P25
animals), but did not observe any major targeting alteration
(Fig. 2C-F). Following this
initial and relatively rough analysis of vomeronasal projections, we
investigated potential finer topographical alterations of the axonal
projection map in this system. Apically located vomeronasal sensory neurons
are known to innervate 15 to 30 glomeruli (with a global position that is
relatively fixed) in each accessory olfactory bulb
(Belluscio et al., 1999;
Rodriguez et al., 1999). To
evaluate putative mis-wiring defects in this system, we made use of a reporter
line we previously generated that allows the identification of axonal
projections of sensory neurons expressing the pheromone receptor gene
V1rb2 (Rodriguez et al.,
1999). No major targeting alteration of V1rb2-expressing
neuron axonal projections was observed in Ac3-null animals
(Fig. 3J,K).
Anteroposterior glomerular shift in Ac3-deficient OSNs
To explain the lack of glomeruli on the caudal part of the main olfactory
bulb, we hypothesized a potential mistargeting of OSNs to inadequate areas, or
alternatively, and more simply, the non-innervation of the bulb by OSNs
expressing specific receptors.
We tested these two possibilities by using three gene-targeted mouse lines,
each allowing the identification of different and defined odorant-expressing
OSNs, and most importantly, of their axonal projections up to their glomerular
targets. We selected the P2-lacZ
(Mombaerts et al., 1996),
MOR23-GFP (Vassalli et
al., 2002) and M72-lacZ
(Feinstein and Mombaerts, 2004)
knock-in lines, corresponding to OSNs located in different neuroepithelial
zones in the nasal cavity, and projecting to very different areas of the main
olfactory bulb.
|
). The
glomerular positions are, as is the rule for mammalian glomeruli, relatively
fixed between animals. In the P2-lacZ reporter line, the
medial glomerulus is readily visualizable on whole-mount views of the medial
side of the bulb (Fig. 3A). No
glomeruli innervated by P2-expressing OSNs were found in Ac3 mutant animals
(nine P15 bulbs analyzed), despite the presence of P2-expressing OSNs sending
axons towards the bulbs (Fig.
3B). In older animals (after P19), a small and weakly labeled
medial glomerulus was rarely observed (data not shown). The global intensity
of the ß-galactosidase staining being weaker in the mutant animals
(Fig. 3A,B), and the staining
intensity being relatively comparable at the single cell level, we compared
the number of P2-expressing OSNs in wild-type and Ac3-/-
turbinates. We counted, using whole mounts, all P2-positive OSNs present on a
well-circumscribed zone: the largest turbinate. As no particular clustering of
P2-expressing OSNs was observed (within their expression zone), we considered
these counts representative of the total number of P2-expressing OSNs. We
found 486±46 and 118±16 OSNs in wild-type and mutant animals
respectively, indicating a drastic reduction of P2-expressing OSNs in
Ac3-/- animals.
Wild-type M72 axonal fibers form two glomeruli per bulb, dorsocaudally
(Fig. 3C,F)
(Potter et al., 2001). Ac3
mutant animals exhibited a very low number of axonal projections at the
topographical coordinates usually chosen by wild-type fibers to generate a
large glomerulus. These projections formed, if any, one to four small
glomerular-like structures (Fig.
3D). This phenotype was observed on both medial and lateral sides
of the bulbs (Fig. 3G).
Strikingly, two large and novel bilaterally symmetrical glomeruli innervated
by M72-expressing axons were observed on the very rostral part of the bulb (21
wild-type and 29 Ac3-/- P15 bulbs analyzed)
(Fig. 3D,E,G;
Fig. 4A,B). The majority of
M72-expressing OSN projections did therefore form two completely novel
glomeruli. Histological analysis of bulbs showed that these M72 glomeruli were
structurally altered and not exclusively innervated by fibers expressing the
M72 receptor (Fig. 5B),
confirming and expanding the results obtained with the rough analysis of
Omp-lacZ Ac3-/- animals. We then counted the
number of M72-expressing OSNs in Ac3-/- and wild-type mice, using
again the largest turbinate as an easily identifiable window. We found
213±51 and 197±40 M72-expressing OSNs in mutant and wild-type
animals, respectively, indicating, in contrast to that observed for the P2
receptor, an unaltered M72-expressing population size.
Axonal projections of wild-type MOR23-expressing OSNs end up on the medial
side of the olfactory bulb, in a centrally located medial glomerulus
(Fig. 3H)
(Vassalli et al., 2002). Ac3
mutant animals exhibited, similarly to the situation observed for M72 axonal
fibers, rare axons reaching the location in the bulb innervated by
MOR23-expressing OSNs in wild-type animals (three P15 bulbs analyzed)
(Fig. 3I). Again, as observed
for M72-expressing OSNs, completely novel glomeruli were formed on the rostral
part of the bulb (Fig. 3I),
receiving input from most of the MOR23-expressing OSN population. These new
glomeruli were not always well defined
(Fig. 3I;
Fig. 5A), and their boundaries
were difficult to identify even histologically due to a loose ensheathing by
periglomerular cells (Fig.
5A).
We plotted the precise position of 121 and 34 glomeruli corresponding to M72- and MOR23-expressing fibers, respectively (Fig. 4A-C). A clear anteroposterior shift was observed for glomeruli innervated by MOR23 and by M72 OSN projections. The positions of the novel glomeruli were conserved between bulbs and between animals, except for dorsomedial M72 glomeruli, which were relatively variable in position (Fig. 4B).
Choice of a glomerulus
Schematically, in wild-type animals, OSNs located in the neuroepithelium of
the nasal septum target to glomeruli located on the medial part of the
olfactory bulb, while those located on turbinates target to the lateral side
of the bulb. We report here that some OSNs lacking Ac3 expression, despite the
innervation of a novel glomerulus by the majority of their apparently
functionally identical neighbors, still project to a location corresponding to
the area innervated by wild-type OSNs. Are there differences between
M72-expressing OSNs targeting to one or other of the glomeruli?
These differences could reside in a variable dependence on Ac3 between OSNs expressing the same odorant receptor gene. Depending on the time (during embryogenesis or later after birth) at which an OSN was born, its Ac3 dependence could be unequal. We did observe the formation of ectopic MOR23 and M72 glomeruli in newborn and adult animals of various ages (Fig. 3D,E), supporting the argument against this hypothesis.
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We then investigated the possibility that some OSNs could be dependent, or independent, on Ac3 expression, a situation that could be reflected by their expression, or non-expression, of Ac3. A clear gradient of Ac3 transcripts was observed by in situ hybridization in the main olfactory neuroepithelium, with apical OSNs more reactive than basal ones (Fig. 1C; Fig. 6A,C). We evaluated Ac3 transcription in OSNs expressing M72 (Fig. 6A-C), MOR23 or P2 in P4-P5 animals. Surprisingly, we found 60 (M72), 71 (MOR23) and 68% (P2) of OSNs in which we were unable to detect Ac3 transcripts (393 MOR23, M72 or P2 OSNs analyzed; Fig. 6D). It should be noted that a lack of signal by in situ hybridization does not necessarily indicate a complete lack of transcription. These Ac3-negative OSNs were mostly located in the most basal part of the neuroepithelium. To investigate the degree of differentiation of these two Ac3 high and low expressors, we performed double in situ hybridizations with an Ac3 probe and a probe recognizing Omp transcripts (which are specifically expressed in mature OSNs) (Fig. 6E,F). We found that over 90% of the Ac3 strongly positive OSNs were also positive for Omp, indicating a relatively advanced degree of differentiation, and suggesting that our identification of two Ac3-expressing populations reflects the maturation stage of OSNs. In conclusion, OSNs expressing an identical receptor represent a non-homogenous population, and can express different levels, or different variants of Ac3 transcripts; but the link, if any, between these two populations and the two targeting options is still lacking.
Lack of neuropilin 1 expression in Ac3-/- OSN projections
Neuropilin 1 is a semaphorin class 3 receptor. A relatively large
population of OSNs projecting to the medial and lateral parts of the olfactory
bulb expresses neuropilin 1 (Schwarting et
al., 2000). Both neuropilin 1 and its ligand Sema-3A have been
reported to play a role in axon guidance processes in the olfactory bulb
(Pasterkamp et al., 1998).
Moreover, a single cell cDNA library from an OSN expressing an olfactory
receptor unable to couple to G proteins was reported to lack neuropilin 1
transcripts (Imai et al.,
2006). We therefore investigated if a potential alteration of
neuropilin 1 expression was associated with the lack of Ac3 expression.
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).
Parasagittal sections of olfactory bulbs indicated that neuropilin 1
expression was completely absent in axonal bundles and nerve layer in
Ac3-/- mice (Fig.
7A,B). Coronal sections through the olfactory bulbs showed a lack
of neuropilin 1 in glomeruli from mutant mice, despite the presence of sensory
neuron projections (Fig.
7D).
We then determined, in wild-type animals, the potential expression of
neuropilin 1 in glomeruli innervated by MOR23-expressing OSNs. A strong
expression was observed in MOR23 axonal fibers forming both lateral
(Fig. 7E-G) and medial
(Fig. 7H-J) glomeruli. The same
analysis was performed with medial and lateral M72 and P2 glomeruli.
Expression of neuropilin 1 was observed in M72 glomeruli
(Fig. 7M,N), whereas none was
found in P2 glomeruli (Fig.
7K,L) (Schwarting et al.,
2004).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Apparently, most members of the olfactory transduction cascade (including
the odorant receptor, the G protein and Ac3) are not only present at the OSN
dendritic endings, which are in contact with the outside world, but are also
observed on the OSN axonal projections, which leave the nasal cavity and enter
the olfactory bulb. In light of this observation, it is naturally tempting to
consider this cascade at the core of the readout mechanism allowing a growth
cone to find an adequate target. The topographical position of a given
glomerulus would thus primarily depend on the odorant receptor it expresses,
as it is possibly the only molecule by which a neuronal population innervating
a given glomerulus differs from the one innervating the neighboring one. This
is to be paralleled with the conclusions of two very recent reports indicating
that expression of a constitutively active Gs subunit or overexpression
of a dominant-negative Pka in OSNs, leads to a shift of glomeruli innervated
by I7-expressing fibers along the anteroposterior axis of the olfactory bulb
(Chesler et al., 2007;
Imai et al., 2006).
If the odorant-induced cascade is involved in axonal guidance mechanisms,
why is Ac3 the only member of the olfactory transduction cascade for which
deletion leads to a drastic modification of the wiring diagram in the
olfactory bulb? First, it is likely that only the first steps of the
odorant-induced transduction cascade (i.e. up to the production of cAMP) are
involved in guidance mechanisms. Second, redundancy in the system could allow,
for example, to compensate for the lack of Golf by the expression of
Gs, a subunit also present in OSNs that is phylogenetically and
functionally (i.e. activating adenylyl cyclases) highly related to
Golf. Such compensatory mechanisms have previously been observed
between G subunits (Offermanns and
Simon, 1998).
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A cautionary note should be added here. If cAMP signals direct all axonal targeting of OSNs, why do we still observe, at least in some parts of the olfactory bulb, apparently relatively unaffected glomeruli? Redundant adenylyl cyclases could again come to the rescue, but it is as likely that other mechanisms independent of cAMP levels are at work.
Ac3-dependent and -independent OSN targeting
Our data show that OSNs expressing an identical odorant receptor (M72 or
MOR23) and lacking Ac3, target to novel, rostrally located glomeruli. However,
a non-negligible and consistent proportion of the OSN population still targets
to topographical positions similar to those observed in wild-type animals
(this does not necessarily mean that these fibers reached this position by
reading the same axon guidance cues that wild-type fibers usually do). How can
we explain this surprising situation?
First, the modification of the bulbar topographical map could reflect the alteration of the upstream map, i.e. the zonal restriction of OSNs in the olfactory epithelium. Our data do not support this hypothesis.
Second, one could imagine that, depending on the position of a given OSN (on the rostroapical part of turbinate 2 versus on the caudoventral portion of another turbinate, for example), the path and the signaling cues that will lead their projections to the bulb are different. Thus, it is possible that for a given OSN, depending on its position, the signal encountered during the wiring process requires or does not require Ac3 function.
Alternatively, one could propose that OSN targeting to the bulb during early development or later is unequally dependent on Ac3, despite the fact that comparison of adult and newborn topographical maps does not show a consistent difference between axonal projection patterns.
Finally, two OSN populations, dependent or not on Ac3 expression, could be constitutively present in the nasal cavity. We found a large proportion of Omp-negative OSNs expressing the odorant receptors M72, P2 or MOR23 containing, if any, very low levels of Ac3 transcripts. As odorant receptor transcription is a sign of OSN relative maturity, one would expect that the expression of molecules involved in guidance mechanisms should precede or be concomitant with odorant receptor expression. Our observations do therefore not rule out the existence of Ac3-dependent and independent wiring requirements for OSNs expressing the same odorant receptor.
Ac3 and glomerular formation
In addition to the glomerular map alterations resulting from the lack of
Ac3, we also report important perturbations in the formation of glomeruli. We
indeed observed glomeruli corresponding to M72 co-innervated by OSNs not
expressing M72, and therefore probably expressing other receptors. This
situation is very different from the one encountered in wild-type animals, in
which glomeruli, even if receiving inputs from OSNs expressing barely
different odorant receptors, do not commingle
(Feinstein and Mombaerts,
2004). The alteration of glomerular formation in Ac3 mutants is
even more pronounced for OSNs expressing the P2 receptor, as they are often
unable to form glomeruli. This could, however, result from a known phenomenon,
termed interdependence: a positive correlation exists between the number of
OSNs expressing a given odorant and the probability of maintaining glomerular
convergence (Ebrahimi and Chess,
2000). As we observed a drastic decrease in the number of
P2-expressing OSNs in Ac3 mutant animals, the inability to form P2 glomeruli
could simply result from a side effect affecting cell numbers.
In conclusion, Ac3 apparently plays a dual role, both in the formation of a functionally homogenous glomerulus, and in its global positioning.
A link between Ac3 and neuropilin 1
We report here a drastic modulation of neuropilin 1 expression in
Ac3-deficient OSN projections. We do not know whether the phenotype we observe
is a direct result of the lack of neuropilin 1. But neuropilin 1 expression
pattern in wild-type animals and experimental evidence indicating its
involvement in OSN guidance mechanisms suggest a functional link between Ac3,
neuropilin 1 and rostrocaudal coordinates in the olfactory bulb. Moreover,
Semaphorin-3A, a chemorepellent protein secreted by ensheathing glial cells in
the nerve layer of the olfactory bulb, is an agonist of neuropilin 1 and is
known to be involved in the targeting of OSNs to the olfactory bulb
(Pasterkamp et al., 1998;
Schwarting et al., 2000;
Taniguchi et al., 2003). A
definitive answer to this question will require a conditional neuropilin
1-null allele, because neuropilin 1 knockout mice die early during
embryogenesis (Takashima et al.,
2002).
As MOR23- and M72-expressing OSNs are neuropilin 1-positive, one may easily
suggest a linear link between the misexpression of Neuropilin1 in Ac3 mutants
and the MOR23 and M72 mis-wiring defects. But then why are neuropilin
1-negative glomeruli, such as P2, also strongly affected by the lack of Ac3?
First, Ac3 function may not only interfere with the expression of neuropilin
1, but also with other guidance cues, still to be identified and potentially
involved in the wiring of P2-expressing fibers. Supporting this explanation,
projections of P2-expressing OSN fibers have been found to be in a
Sema-3A-/- background abnormally distributed in the bulb and
converging to atypical places (Schwarting
et al., 2004; Taniguchi et
al., 2003). This phenotype, different from the one we report here
in Ac3-null mice, supports the idea that our observations do not result
exclusively from neuropilin 1 inhibition. Second, considering the dramatic
shift of a large number of glomeruli towards the rostral parts of the bulb,
the position and possibly the establishment of glomeruli that would normally
be located at this position will probably be affected. Thus, a possible
competition for permissive target areas may take place in the rostral parts of
the bulb, and explain the indirect mistargeting of OSN fibers pertaining to
OSNs not expressing neuropilin 1. Third, as discussed previously, the lack of
glomerular formation by Ac3-/- P2 fibers may simply reflect a too
low number of P2-expressing neurons, and therefore not represent, unlike MOR23
and M72 projections, an adequate tool to study the role of Ac3 in guidance
mechanisms.
|
), but
large gaps are still remaining. Parallels between signaling pathways that
allow growth cones to adequately react to their surroundings, and those
involved in guidance of non-neuronal structures such as vascular networks, for
example (Carmeliet and Tessier-Lavigne,
2005), have already emerged. These, including the recognition of
cyclic nucleotides as key elements in transduction cascades, will surely soon
be expanded, and provide a integrative understanding of neuronal guidance
mechanisms. |
ACKNOWLEDGMENTS |
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|
Footnotes |
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