As mentioned previously, TrpC2 is an ion channel supposedly exclusively present in vomeronasal sensory neurons. It is crucial for VR induced signal transduction.
Its general deletion in mice leads to striking behavioral modifications that include a lack of gender discrimination, an observation that led to the conclusion that the vomeronasal organ was responsible for this latter task. However, the physical removal of this structure does not replicate the behavioral modifications observed in the mutant animals.
Preliminary observations in the laboratory pointed to the existence of non-vomeronasal olfactory sensory neurons that also express TrpC2, an observation possibly explaining the behavioral discrepancies between animals with a “genetically ablated” vomeronasal organ and those whose organ was physically removed.
We here explore further the potential existence of a neuronal population expressing TrpC2 in the main olfactory epithelium.
Transient Expression of TrpC2 in the Main Olfactory Epithelium
We evaluated the expression of TrpC2 in OSNs by immunohistochemistry on coronal sections of the main olfactory epithelium. We analyzed young animals (PND0, PND7, PND21) and adults.
In the main olfactory epithelium, we found numerous OSNs labeled in PND0 (13.5% of the OSNs: 283/2093), and PND7 (5.3%; 205/3861 OSNs) mice (Figure 33). These numbers further decreased at PND21 (1.5%; 161/10700 OSNs) to virtually disappear in adults (data not shown). These results are in agreement with a previous report showing almost inexistent TrpC2 expression in adult rat OSNs (Liman et al., 1999).
The morphology of the TrpC2-expressing OSNs was similar to the one of OR gene-expressing OSNs (Figure 34A).
Figure 33: Coronal section of the main olfactory epithelium of newborn (A), PND7 (B) and PND21 (C) mice. With age, a decrease in TrpC2-expressing OSNs is observed. Inserts represent corresponding vomeronasal organ sections. PND: postnatal day. Scale bars: 100μm.
In order to link these OSNs to a known subpopulation of the main olfactory epithelium, we characterized these cells more precisely by evaluating their expression of specific OSN markers. By immunohistochemistry, we tested OMP (a marker for mature OSNs), and markers specific for two subpopulations of OSNs located in the main olfactory epithelium: IP3 receptor 350, a marker for Jourdan cells, and carbonic anhydrase type 2, a marker for GC-D cells. In PND7 mice, no TRPC2-positive OSNs were also IP3 receptor 3- or carbonic anhydrase type 2-positive (Figure 34B,C), arguing against a Jourdan or GC-D cell identity of the TrpC2-expressing OSNs. Surprisingly, most of the TRPC2-positive OSNs were negative for OMP (Figure 34A) despite their location in the upper part of the neuroepithelium.
Figure 34: Immunohistochemistry on main olfactory epithelium coronal sections of PND7 mice.
The majority of TRPC2-positive OSNs are OMP-negative (A, the asterisk indicate a double-positive OSN). TrpC2-positive OSNs are negative for IP3 receptor 3 (B) and carbonic anhydrase type 2 (C). CA2: carbonic anhydrase type 2, ITPR3: IP3 receptor 3, OMP: olfactory marker protein, TRPC2: transient potential channel C2, Scale bars: 100μm.
This population thus does not appear to share markers known to characterize OSNs in the main olfactory epithelium.
50 The Itpr3 gene, also known as Ip3r3, codes for the IP3 receptor 3 protein.
Permanent Labeling of TrpC2-expressing OSNs
We then asked the following question: Are the TrpC2-expressing OSNs a population that disappears with sexual maturity or are these OSNs maintained in adulthood but loose their expression of TrpC2? To answer this, we opted for a transgenic approach.
The TrpC2 promoter is not characterized; we thus generated transgenic mouse lines by using a BAC containing the TrpC2 gene. We inserted an IRES-Cre -IRES-GFP cassette immediately after the TrpC2 stop codon (Figure 38, see methods).
The GFP coding sequence was to permit the direct visualization of the cells expressing the transgene, while the expression of Cre was to allow for the conditional activation of a remote coding sequence (like for instance, a permanently expressed reporter).
We generated eight independent TrpC2-IRES-Cre-IRES-GFP transgenic mouse lines, all of which transmitted the transgene.
To visualize all cells that transiently expressed TrpC2, we crossed the BAC transgenic mice with reporter lines constitutively expressing lacZ (Rosa26+/(stop)lacZ) or RFP (Rosa26+/(stop)RFP) upon Cre-dependent removal of a stop cassette (Luche et al., 2007; Soriano, 1999). We analyzed adult animals.
As expected, seven lines expressed strongly the reporter in the vomeronasal organ (Figure 36A,D and data not shown). The remaining line exhibited reporter expression in many tissues, including the whole brain, most other organs and the skin. This line, probably representing an aberrant very early expression of the transgene during development, was thus discarded.
Similarly, all seven lines showed very similar patterns in the main olfactory epithelium, although the number of labeled OSNs differed. In all lines, labeled cells were located mostly apically in the neuroepithelium (Figure 35 and data not shown), and were found homogeneously scattered on the surface of this latter (no zonal restriction was observed). We chose to focus on two lines (4693 and 4700) because they represented respectively high and low expressors of the transgene (Figure 35).
Characterisation of the TrpC2 Expressors
We first tested the potential coesxpression of TrpC2 and Omp, the main marker of mature OSNs. We thus crossed TrpC2-IRES-Cre-IRES-GFP; Rosa26+/(stop)RFP transgenic animals (where TrpC2 expression leads to permanent RFP expression), with Omp-GFP mice (in which all mature OSNs are labeled). We performed two-color immunohistochemistry on main olfactory epithelium sections to detect RFP and GFP. We did use the GFP fluorescence from the Omp-GFP line since our TrpC2-IRES-Cre-IRES-GFP transgenics exhibited no
endogenous expression of GFP, or in the case of the high expressor line, weak expression in VSNs only (data not shown).
We found little overlap between OMP-positive and RFP-positive cells (Figure 35). These experiments confirmed that most RFP-positive cells were located in the apical part of the main olfactory epithelium. These cells were either OMP-negative or weakly positive. This phenotype seems to indicate some kind of exclusion between cells that once expressed TrpC2 and cells that express Omp.
Figure 35: Immunohistochemistry on coronal sections of the main olfactory epithelium of adult mice. TrpC2 expressors (red) are clearly apically located in the main olfactory epithelium. A majority of these cells are negative for OMP. Sections of the high expressor (A,B) and the low expressor (C,D) lines are shown. Scale bars: 100μm
Axonal Projections of TrpC2-expressing OSNs
We then took advantage of the TrpC2-IRES-Cre-IRES-GFP; Rosa26+/(stop)RFP mouse lines to evaluate the projection pattern of the OSNs that did express TrpC2. In order to visualize the all glomeruli present in the bulb, we crossed this line, again with the Omp-GFP line. A dorsal view of the corresponding animal showed that only a limited area of the bulb was consistently innervated by RFP positive neurons (Figure 36). This area was located in the very rostromedial part of the bulb, and did include about 40 glomeruli. The seven initial transgenic lines did exhibit convergence in this rostromedial part of the bulb. Four of these lines (including line 4693) where also characterized by an additional diffuse axonal targeting in other parts of the bulb.
Figure 36: Projections of TrpC2-expressing OSNs (red). Whole mount views of the high (A-C) and low (D-F) expressor lines are shown. The accessory olfactory bulb and the rostromedial part of the main olfactory bulb are labeled in both transgenic lines. The green fluorescence corresponds to OSNs that are positive for the OMP marker.
Discussion
Study 1: Olfactory Transduction and Axonal Guidance
cAMP as a Major Player in OSN Axonal Guidance
Our experiments aimed at the identification of the cues that allow OSNs to find their correct targets in the olfactory bulb. As previously indicated, reports suggest that the expressed olfactory receptor plays a role in this process (Feinstein et al., 2004; Mombaerts et al., 1996; Rodriguez et al., 1999; Wang et al., 1998)
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. 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. This is to be paralleled with the conclusions of two reports indicating that the alteration of the expression of members of this cascade, affects OSN guidance. Indeed, a constitutively active Gαs subunit or the overexpression of a dominant-negative protein kinase A 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).
We found various alterations of olfactory guidance processes in mice lacking AC3, alterations that will be discussed in the two following sections. 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 topographical map in the olfactory bulb? First, it is possible 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 Gαolf by the expression of Gαs, a subunit present in immature OSNs that is phylogenetically and functionally (i.e. activating adenylyl cyclases) highly related to Gαolf. Such compensatory mechanisms have previously been observed between Gα subunits (Offermanns and Simon, 1998).
The same logic of redundancy could be applied to our inability to find wiring defects in VSN projections. Indeed, in this last tissue, adenylyl cyclase 2 is highly expressed (Berghard and Buck, 1996).
We know now that the expression of genes involved in axon guidance is correlated with the expression of a given odorant receptor gene in OSNs.
However, at the beginning of our study, the potential link between these data was not known and it remains today poorly characterized.
AC3-dependent and -independent OSN Targeting
Our data show that OSNs expressing a given odorant receptor gene (M72 or MOR23) and lacking Adcy3, 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 control 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 direct their projections towards 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 Adcy3 expression, could be constitutively present in the nasal cavity. We found that a large proportion of OMP-negative OSNs expressing the odorant receptors gene M72, P2 or MOR23 contain, if any, very low levels of Adcy3 transcripts. As OR 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 OR gene expression. Our observations do therefore not rule out the existence of AC3-dependent and independent wiring requirements for OSNs expressing the same OR gene.
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 coinnervated by OSNs not expressing M72, and therefore probably expressing other OR genes. This situation is very different from the one encountered in control 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 Adcy3 mutants is even more pronounced for OSNs expressing P2, as young animals 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 Adcy3 mutants, the delayed formation of P2 glomeruli could simply result from a side effect affecting cell numbers.
In conclusion, in axon guidance AC3 apparently plays a dual role , both in the formation of a functionally homogenous glomerulus, and in its global positioning.
Adcy3 and Negative Feedback
We hypothesized that non-homogenous innervation of M72 glomeruli could reflect the coexpression of OR genes, and therefore a compound guidance code that would result in an alteration of the glomerular homogeneity. We tested this hypothesis by evaluating the potential coexpression of Mor28 and olfr727 in Adcy3 mutant OSNs. The probability of these gene to be chosen is high, and the OR pair is located in the same sensory zones. No coexpression was observed between Mor28 and olfr727, strongly arguing against a role of AC3 in negative feedback.
An alternative hypothesis, linked to the negative feedback, has however not been ruled out by our experiments. AC3 activity, or the OR-driven signal, could lead to a prevention of early OR gene switching. This switching event is observed for some ORs (Shykind et al., 2004), but not for others (Li et al., 2004). Allowing OR switching should not necessarily result in multiple ORs expressed at a time. But if it takes place after OSNs have reached their glomerular targets, it would likely lead to an apparently altered topographical map.
For example, among the M72-expressing population, there would be those that have not switched from their initial OR choice, and thus keep expressing M72. These would target to a M72 glomerulus and be visible on an M72-LacZ background. Those that have switched from M72 to another OR gene would not be visible anymore on the same background. These would still innervate M72 glomeruli but not express β-galactosidase anymore. Finally, those that would have initially chosen an OR gene and would later (after targeting to their glomeruli) switch to M72, would look like stray blue fibers, targeting apparently away from their endogenous glomerulus. This hypothetical phenotype is in fact strikingly similar to the one we observe for M72 fibers in Adcy3 mutant animals.
To evaluate this hypothesis, we are currently using a mouse line that drives the expression of the Cre recombinase under the control of the M71 gene (an OR gene very closely related to M72, both in sequence and in genomic location). All
OSNs that once expressed M71 can therefore be labeled with the use of a reporter. If the hypothesis is correct we expect that a) a fraction of these labeled OSNs, in an Adcy3 mutant background, now express other ORs and b) that the M71 glomerulus that maintains its endogenous position in the Adcy3 mutant, is now exclusively innervated by fibers that emanate from OSNs that express, or expressed once M71.
AC3-dependent Genes
As mentioned repeatedly, our hypothesis was that a signal, mediated by the expression/activation of the OR receptor, was somehow involved in guiding olfactory axons. We hypothesized that the perturbation of the olfactory cascade, namely the prevention of Adcy3 expression, would affect this wiring, and likely be associated with some subtle alterations of the olfactory transcriptome.
The expected limited transcriptome alteration turned out to be a massive one.
Although multiple genes involved in guidance processes were up or downregulated in OSNs from Adcy3 mutants, and although some of them were possibly involved in the guidance phenotype observed in thesemutants, we did not consider worth pursuing each of them separately.
Our DNA chip experiment indicates first that AC3 plays a crucial role in OSNs, possibly in addition to its known function in OR transduction. Second, despite the misexpression of over a dozen genes coding for guidance molecules, a fraction of Adcy3 mutant OSNs are still able to coalesce and form glomeruli, suggesting that the mechanism that drives this convergence is particularly robust.
A Link Between AC3 and NRP1
We found a drastic modulation of NRP1 expression in Adcy3-deficient OSN projections. Given the numerous genes (including some coding for guidance molecules) whose expression was perturbed in these mutant mice, we thought that the guidance phenotype we observed was probably not solely due to the lack of NRP1. Mutants lacking specific guidance molecules such as SEMA3A have indeed been shown to exhibit altered olfactory projections sites (Schwarting et al., 2004).
In addition to this general and obvious remark, a few precise observations argue against the lack of NRP1 as the sole responsible for the AC3 phenotype.
First, as MOR23- and M72-expressing OSNs are NRP1-positive, a link between the misexpression of Nrp1 in Adcy3 mutants and the mis-wiring defects observed for OSNs expressing these OR genes is easy to make. But then why are NRP1-negative glomeruli, such as P2, also strongly affected by the lack of AC3?
However, if we consider the dramatic shift of a large number of glomeruli
towards the rostral parts of the main olfactory bulb, the position and possibly the establishment of glomeruli that would normally be located at this position should 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 Nrp1.
Second, as discussed previously, the delayed glomerular formation by Adcy3-/- P2 fibers may simply reflect a too low number of P2-expressing OSNs, and therefore, unlike MOR23 and M72 projections, not represent an adequate tool to study the role of AC3 in guidance mechanisms.
Given the peculiar expression pattern of NRP1 in the olfactory system and despite these observations (that argue against focusing on NRP1), we investigated its role in olfactory axonal guidance. And to this aim, we analyzed conditional Nrp1 mutant mice.
NRP1-dependent OSN Targeting
Our data show that OSNs expressing a given odorant receptor gene (M71 or M72) and lacking Nrp1, target to glomeruli that are medially shifted (compared to controls), resulting in glomerular fusion in the case of M71 and glomerular interconnection in the case of M72. How can we explain these phenotypes?
First, these two phenotypes may reflect a similar effect on two OSN populations, one of them being simply exacerbated because of the nature of the ORs expressed by these populations. Since the identity of the OR gene expressed by an OSN correlates with the expression of specific sets of guidance genes (Cutforth et al., 2003; Serizawa et al., 2006), different OSN subpopulations could rely unequally on NRP1.
Second, the different M71 and M72 Nrp1-deficient phenotypes may reflect the timing at which OSNs are depleted of NRP1, in other words at what time the Nrp1 coding sequence is removed. Indeed, in M71-expressing OSNs the Cre-mediated Nrp1 excision is concomitant with the transcription of the OR
Second, the different M71 and M72 Nrp1-deficient phenotypes may reflect the timing at which OSNs are depleted of NRP1, in other words at what time the Nrp1 coding sequence is removed. Indeed, in M71-expressing OSNs the Cre-mediated Nrp1 excision is concomitant with the transcription of the OR