Negative Feedback or Not?

Dans le document Vomeronasal receptors: from monogenic expression to axon guidance (Page 97-0)

As discussed and shown previously, the expression of an OR in vomeronasal neurons leads to the silencing of all V1Rs. We were naturally expecting that the overexpression of V1rb2 in most sensory neurons of the main olfactory system would block the expression of odorant receptor genes.

To answer this question, we crossed the OMP-D-V1rb2#2 line with two OR knock-in lines, which allow an easy visualization of OSNs expressing the M71 and P2 OR genes. Expression of both ORs was not altered despite the cotranscription of the V1rb2 transgene (data not shown), indicating that the expression of the transgene did not induce a negative feedback on the transcription of ORs.

line

#4 line

#1 MOE

V1rb2glyc

*

pA OMP

VNO

wt

OMPCre wt

OMPCre

A

C

E

G

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OMP-D-V1rb2glyc

Figure 6.17: Overexpression of V1rb2 in OMP-D-V1rb2glyc mouse lines. In situ hy-bridizations of coronal sections of the head of mice belonging to 2 different OMP-D-V1rb2glyc mouse lines. Overexpression of V1rb2 is clearly visible in both the MOE and the VNO in absence of the Cre recombinase. In the VNO, overexpression is limited to the basal layer, where V2Rs and M10/M1 MHC families are expressed (B,F). Strong punc-tate labelling in the VNO corresponds to the endogenous expression ofV1rb2. Scale bar, 200µm.

6.3.5 Global Glomerular Map in Animals Overexpress-ing V1rb2

Axonal projections in the olfactory bulb depend on the expression of odorant and vomeronasal receptors by sensory neurons. Having in hands a mouse expressing a V1R in most olfactory sensory neurons, we wondered if the topographical map in the bulb was affected.

We crossed theOMP-D-V1rb2#2 line with theOMP-taulacZ line (Potter

et al., 2001). This latter line allows to easily follow all axonal projections in the olfactory bulb. Comparison of 2-week-old transgenic mice showed no obvious differences with wild-type animals (Figure 6.18 on page 84). A similar observation was made on 8.5-week-old mice.

E

C

P15 P60

B D

A

lacZ

V1rb2pAOMP wt

OMP tau::lacZ

H

F G

Figure 6.18: Axonal projections in OMP-D-V1rb2 × OMP-taulacZ mice. No obvious alteration of the bulbar topographical map was observed inOMP-D-V1rb2 mice. Dorso-posterior view of OB fromOMP-taulacZ mice. Analyses were performed on P15 and P60 mice. Scale bar, 400µm.

6.3.6 Glomerular Targeting in Animals Overexpress-ing V1rb2

OSNs expressing a given OR do project to two glomeruli per bulb, at a rela-tively stereotyped place. To perform a more precise analysis of the potential targeting alterations induced by the expression of theOMP-D-V1rb2 trans-gene, we took advantage of knock-in lines which allow an easy visualization of axonal projections in the olfactory bulb.

The two lines previously used for the monogenic evaluation were cho-sen: theM72-M72-taulacZ and P2-P2-taulacZ reporter lines (Feinstein and Mombaerts, 2004; Mombaerts et al., 1996). Their projection patterns are very different, one characterized by axons projecting very close to the crib-riform plate and the other very dorsally. We analysed the MOE and OB of mice at different time points of development, starting at one week.

Effect of V1rb2 Overexpression on M72-expressing OSNs

We performed X-Gal whole-mount stainings on M72-M72-taulacZ × OMP-D-V1rb2 littermates. To evaluate a potential alteration in the number of OSNs expressing M72, we counted all lacZ-positive neurons from a defined zone in the turbinates (depicted in Figure 6.19A on page 86).

In wild type animals, we scored 45.83 ±27.43 (n = 12 MOEs) and 63.45

± 20.82 (n = 11 MOEs) M72-expressing OSNs for 1–2-week-old and 3–5-week-old animals, respectively. In OMP-D-V1rb2 mice, we found 54.07 ± 26.67 (n = 27 MOEs) and 88.80 ± 32.46 (n = 10 MOEs) OSNs expressing the lacZ marker, respectively (Figure 6.19 on page 86). A Student’s t-Test showed no significant difference for aP < 0.01.

We also evaluated a potential shift in the MOE zone whereM72-expressing OSNs are located. This was a necessary control, since it is known that the topography of axonal projections is dependent on the MOE zone from which the axons emanate. No obvious alteration of the expression zone ofM72 was observed in OMP-D-V1rb2 transgenic mice (Figure 6.19 on page 86).

140 120 100 80 60 40 20 1–2wo 3–5wo 0

G number of M72-expressing OSNs

1–2wo 3–5wo

**

V1rb2 pA OMP

M72 M72 IRES tau::lacZ

wt

A lacZ

C

E

B

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Figure 6.19: V1rb2 overexpression in the MOE. (A-F) Lateral views of M72-M72-taulacZ mice, wild-type (A,C,E) or expressing the OMP-D-V1rb2 transgene (B,D,F).

(G) LacZ-expressing OSNs in the region corresponding to the dotted area in (A). Each dot corresponds to the number of lacZ-positive OSNs. The horizontal bar is the overall mean. The vertical bar represents the standard deviation. ** = P < 0.05. Scale bar, 300µm.

M72 Projections towards the OB in Animals OverexpressingV1rb2

We then analysed the projections of M72-expressing OSNs in an OMP-D-V1rb2 background. As mentioned, in wild-type animals, OSNs expressing M72 project their axons to a specific zone of the OB and form two, one medial and one lateral glomerulus. The presence of the OMP-D-V1rb2 transgene drastically affected this pattern. Many were apparently “lost” or coalesced into multiple glomeruli (Figure 6.20 on page 87).

wt

V1rb2pAOMP

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A

lacZ

B C D

M72 M72 IRES tau::lacZ

Figure 6.20: Projections towards the olfactory bulb inOMP-D-V1rb2 mice. Dorsopos-terior view ofM72-M72-taulacZ olfactory bulbs. Axons emanating from M72-expressing OSNs in aOMP-D-V1rb2 background coalesce into multiple glomeruli (E-H) unlike what is observed in the wild-type situation (A-D). Scale bar, 400µm.

The number of M72 glomeruli was counted to evaluate the degree of perturbation caused by theOMP-D-V1rb2 construction. We confirmed that

in wild-type mice each OB contains one medial and one lateral glomerulus (although with slight variations). We counted 1.41 ± 0.56 (n = 18 bulbs), 1.08±0.29 (n = 6 bulbs) and 1.00±0.00 (n = 6 bulbs) medial glomeruli for 1–2-, 3–5- and 6–8-week-old animals, respectively (Figure 6.21C on page 89).

The numbers of lateral glomeruli for age-matched animals were the following:

1.26 ± 0.51 (n = 35 bulbs), 1.25 ± 0.45 (n = 12 bulbs) and 1.08 ± 0.29 (n

= 12 bulbs) glomeruli, respectively (Figure 6.21D on page 89).

In OMP-D-V1rb2 mice, we counted 3.50 ± 2.47 (n = 8 bulbs), 2.54 ± 1.45 (n = 7 bulbs) and 2.75 ± 1.22 (n = 6 bulbs) medial glomeruli for 1–2-, 3–5- and 6–8-week-old animals, respectively (Figure 6.21C on page 89). The number of lateral glomeruli was 2.21± 1.58 (n = 14 bulbs), 2.93 ± 1.59 (n

= 14 bulbs) and 2.00 ± 0.60 (n = 12 bulbs), respectively (Figure 6.21D on page 89). A Student’st-Test confirmed that these differences were significant:

allPs were inferior to 0.01, except for the value corresponding to the medial glomeruli in 1–2-week-old animals, where P < 0.05.

Effect of V1rb2 Overexpression on P2-expressing OSNs

We then studied the effect of V1rb2 overexpression on the targeting of an-other OR, P2. We used the P2-P2-taulacZ mouse line, in which the class II P2 OR is coupled with the taulacZ marker.

We first evaluated if the zone in the MOE where P2 is expressed was altered on theOMP-D-V1rb2 background. No alteration was observed (data not shown).

Comparing P2 axonal projections in wild-type and OMP-D-V1rb2 ani-mals, we found no difference with respect to the coordinates in the OB where axons fromP2-expressing OSNs coalesce (Figure 6.22 on page 90). We how-ever found that, like what we observed for M72, the number of P2 glomeruli varies in a significant way. The number of medial glomeruli was 1.12 ±0.33 (n = 33 bulbs) in wild-type animals and 1.86 ± 0.88 (n = 29 bulbs) in OMP-D-V1rb2-expressing mice (Student’s t-Test, df = 60, P <0.01).

Taken together, our data indicate that the expression of V1rb2 alters

V1rb2 pA OMP

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number of medial glomeruli

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8 6 4 2

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* *

Figure 6.21: Altered M72 glomerular map in OMP-D-V1rb2 mice. (A-B) The lateral topographical map for M72 is heavily disrupted in OMP-D-V1rb2 mice. (C-D) In mice overexpressing V1RB2, axons coming fromM72 OSNs coalesce into more than one medial and one lateral glomeruli. Each dot corresponds to the number of glomeruli present in an OB. The horizontal bar is the overall mean. The vertical bar represents the standard deviation. * =P <0.01, ** =P <0.05. Scale bar, 300µm.

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P2 P2 IRES tau::lacZ

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Figure 6.22: Altered number of P2 glomeruli in OMP-D-V1rb2-expressing mice. (A-B) Projections of P2-expressing OSNs in wild-type (A) and OMP-D-V1rb2-transgenic animals (B). (C) Schematic showing the position of all P2 glomeruli counted. (D) OMP-D-V1rb2-expressing animals have a larger number of glomeruli compared to age-matched wild-type animals. Each dot corresponds to the number of glomeruli present in an OB.

The horizontal bar is the overall mean. The vertical bar represents the standard deviation.

P <0.01. Scale bar, 300µm.

significantly axonal projections in the main olfactory bulb.

6.3.7 Discussion, Part III

Our results first indicate that forcing the expression of a given olfactory recep-tor in the olfacrecep-tory system is not an easy task. Multiple transgenic attempts to overexpress V1rb2 did fail. This even using different promoters which were active before or after endogenous olfactory gene choice, and even whose activation was inducible. A mechanism somehow silences the transgenes.

We only found a single way to override this mechanism. But we do not know exactly what we did to make it right. Is it the physical separation between theV1rb2 CDS and the promoter driving its expression which allows

the receptor to be expressed? This view would be compatible with the fact that after removal of the stop cassette, no more transcription of the transgene is observed. The NSE-CreERT2-V1rb2 -DsRed2 and Gγ8 CreERT2V1rb2 -DsRed2 transgenes also posses a V1rb2 CDS separated from the promoter.

Maybe that what it takes is an olfactory type promoter like OMP, and a distance between this promoter and the V1rb2 CDS.

We should keep in mind that OMP, and therefore the transgene, is active only after OR or V1R promoters are active. This means that we do not know if the “negative feedback” acting of the transgene after removal of the stop cassette (or distance cassette) is driven by the endogenous and already expressed chemoreceptor, or by a general mechanism which keeps ORs and V1Rs usually silent.

No V1rb2-driven negative feedback was observed on endogenously ex-pressed ORs. Does that mean that V1rb2 is unable to prevent expression of ORs? Likely not, since ORs prevent the expression of V1Rs and since a swap of V1rb2 in the M71 locus leads to axonal convergence (suggesting monogenic expression) (Feinstein et al., 2004). It possibly reflects the late expression of the transgene, which is active, again, after endogenous chemore-ceptor genes are chosen for expression. But in this case, why is the OMP transgene working at all? If negative feedback exists, should it not silence the transgene? As discussed earlier, maybe that this is exactly what we see when we remove the “distance cassette”. When the promoter is close to the V1rb2 CDS, negative feedback can take place. Separating the promoter and the CDS prevents this negative feedback.

It should be noted that two relatively recent studies reported successful overexpression of ORs in the main olfactory system. Both were based on an indirect approach, based on a tetracycline-transactivator (TTA/tetO) sys-tem. In this approach, the olfactory promoter was physically separated from the OR CDS (Nguyen et al., 2007; Fleischmann et al., 2008). No mechanis-tic insight was provided by these publications, but this sounds reminiscent of our results. So a signal is likely present in chemoreceptor CDSs.

Significant alterations of axonal projection patterns were observed in V1rb2-overexpressing lines. Multiple M72 and P2 glomeruli were found on each side of the bulbs. What does that mean? Does the expression of V1RB2 together with an OR provide a confusing code to axons? Or is it that the peculiar projection patterns observed in the accessory olfactory bulb in wild type animals (i.e. the presence of multiple glomeruli corresponding to a single V1R) is an intrinsic information embedded into V1R receptors, that is simply revealed here?

Perspectives

Our results are relatively challenging. In particular the ability of a 7TM, rhodopsin, to apparently substitute for axon guidance and negative feedback processes. Are the apparent unique characteristics we observe in the olfactory system just the tip of an iceberg? Are other neural systems using the same types of mechanisms?

This may be. But even if it is not, the problem we face in the olfactory system is remarkable. It represents a beautiful biological question. How to express a single chemoreceptor gene among a choice of over 2000? And how to wire the axon corresponding to this choice to the right place in the brain?

These two questions have been asked a while ago. In fact over 15 years ago.

And nobody has provided a satisfactory answer yet.

I bet that the mechanisms used to achieve this formidable task are dif-ferent from the ones we are used to handle, and that this is why they hide a little.

Transgenic Mouse Lines

Figures A.1 and A.2 on pages 95–96 depict all transgenic mouse lines used in this thesis, with the indication of the canonical name according to the “MGI Guidelines” available at the Jackson Laboratory website (<http://www.informatics.jax.org/mgihome/nomen/gene.shtml>) and the common name our laboratory and others use.

Tg(GS129-VR2)1Ugfs V1rb2-V1rb2-taulacZ

V1rb2 V1rb2 IRES tau::lacZ tm1Mom

V1rb2 V1rb2 vt

V1rb2 V1rb2 IRES tau::lacZ

V1rb2 GFP IRES tau::lacZ

tm3Mom

V1rb2 V1rb2 dv

V1rb2 Rho IRES tau::lacZ

Tg(RP24-167L24)6Ugfs V1rb2-Rho-taulacZ

tm2Mom

V1rb2 V1rb2 vg

V1rb2 V1rb2 IRES tau::GFP

V1rb2 M71 IRES tau::lacZ

tm4(Olfr151)Mom

V1rb2 V1rb2 mv

Figure A.1: Mouse lines used in theV1rb2mvandV1rb2-Rho-taulacZ projects.

tm1Mom

Figure A.2: Mouse lines used in theV1rb2 overexpression project.

COMMENTARY

Non-exclusive exclusion

(Commentary on Capello et al.)

Diego Rodriguez-GilandCharles A. Greer

Departments of Neurosurgery and Neurobiology, Yale University School of Medicine, New Haven, CT 06520, USA

Beginning with their discovery by Buck & Axel (1991), a series of significant advances mark our increased understanding of odor receptors (ORs) and their roles in both odor transduction and axon coalesence (Imai & Sakano, 2008). Somewhat unexpectedly, the family of 1200+ ORs (Zhang &

Firestein, 2002) found in the main olfactory epithelium exhibit little homology to those found in the vomeronasal organ where two families, of over 300 vomeronasal receptors (VRs) (Touhara, 2008) mediate pheromone detection. They differ in many respects, including the downstream transduction cascade, but share the elegant property of allelic exclusion, which results in any one sensory neuron expressing one OR or VR from either the maternal or paternal chromosome, but not both. While the significance of allelic exclusion in processing sensory information in the olfactory and vomeronasal systems remains unknown, the mechanisms regulating allelic exclusion are of intense interest.

In this issue, Capelloet al.(2009) address the question of whether mechanisms of allelic exclusion are similar in both the olfactory and vomeronasal sensory systems. They approached the question using a mouse model in which the odorant receptor M71, normally expressed in the main olfactory epithelium, is now expressed in the vomeronasal organ. This was achieved by using the promoter region of the vomeronasal gene V1RB2 to direct the expression of M71 in vomeronasal sensory neurons in lieu of V1RB2 expression. It was advantageous that M71 has a low percentage of identity with V1RB2 protein, or with any member of the V1R family of vomeronasal receptors.

Previous results showed that axons from new M71 > V1rb2 neurons target the accessory olfactory bulb where they coalesce to form homogenous glomeruli (Rodriguezet al., 1999). Here, Capelloet al.(2009) show that these neurons do not express the endogenous V1RB2 receptor or any member of the V1R family of genes. Because members of the odor transduction cascade are important in axon targeting from the main olfactory epithelium (Chesleret al., 2007) and, because the transduction signaling molecules used by main olfactory and vomeronasal sensory neurons are different, the authors then ask whether the expression of M71 also changes the expression of members of the transduction cascade. Vomeronasal sensory neurons express Galphai2 and TRPC2, while olfactory sensory neurons express Galphaolf, AC3 and CNGA2.

Interestingly,M71 > V1rb2neurons continue to express markers characteristic of vomeronasal neurons; it appears that their essential identity or phenotype is unchanged other than the expression of M71 in lieu of V1RB2.

Capelloet al.(2009) show, for the first time, that allelic exclusion in vomeronasal receptor cells can occur through the expression of an exogenous receptor and, that exclusion of expression is not limited to the substituted receptor, but applies to all members of the family – exclusion which is not exclusive. It is tempting to speculate that allelic exclusion may be regulated by universal mechanisms, as suggested by Cedar & Bergman (2008) in considering antigen receptor expression in the immune system. However, the nature of the cascade mediating the one receptor - one sensory neuron rule in the main olfactory epithelium and the vomeronasal system remains elusive. While Capelloet al.(2009) have shown that M71 effectively excludes expression of V1Rs, and that the axons coalesce appropriately into accessory olfactory bulb glomeruli, the reciprocal replacement is not effective;V1rb2>M71does not result in viable olfactory sensory neurons whose axons converge in the main olfactory bulb (Feinsteinet al., 2004).

Contributing to the challenge of understanding these mechanisms in both of these sensory systems is the size of the receptor families and understanding other molecular determinants of differentiation and axon targeting in main olfactory and vomeronasal sensory neurons. Despite many unanswered questions, the current paper from Capelloet al.(2009) provides a new perspective from which to address the mechanism(s) of allelic exclusion, odorpheromone transduction, and axon targeting. To paraphrase Shakespeare, would a rose by any other receptor smell as sweet?

References

Buck, L. & Axel, R. (1991) A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.Cell,65, 175–187.

Capello, L., Roppolo, D., Pauli Jungo, V., Feinstein, P. & RodrI`guez, I. (2009) A common gene exclusion mechanism used by two chemosensory systems.Eur. J.

Neurosci.,29, 671–678.

Cedar, H. & Bergman, Y. (2008) Choreography of Ig allelic exclusion.Curr. Opin. Immunol.,20, 308–317.

Chesler, A.T., Zou, D.J., Le Pichon, C.E., Peterlin, Z.A., Matthews, G.A., Pei, X., Miller, M.C. & Firestein, S. (2007) AG proteincAMP signal cascade is required for axonal convergence into olfactory glomeruli.Proc. Natl. Acad. Sci. USA,104, 1039–1044.

Feinstein, P., Bozza, T., Rodriguez, I., Vassalli, A. & Mombaerts, P. (2004) Axon guidance of mouse olfactory sensory neurons by odorant receptors and the beta2 adrenergic receptor.Cell,117, 833–846.

Imai, T. & Sakano, H. (2008) Odorant receptor-mediated signaling in the mouse.Curr. Opin. Neurobiol.,18, 251–260.

Rodriguez, I., Feinstein, P. & Mombaerts, P. (1999) Variable patterns of axonal projections of sensory neurons in the mouse vomeronasal system.Cell,97, 199–208.

Touhara, K. (2008) Sexual communication via peptide and protein pheromones.Curr. Opin. Pharmacol.,8, 759–764.

Zhang, X. & Firestein, S. (2002) The olfactory receptor gene superfamily of the mouse.Nat. Neurosci.,5, 124–133.

Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.

European Journal of Neuroscience, p. 1, 2009 doi:10.1111/j.1460-9568.2009.06658.x

ªThe Authors (2009). Journal CompilationªFederation of European Neuroscience Societies and Blackwell Publishing Ltd

MOLECULAR AND DEVELOPMENTAL NEUROSCIENCE

A common gene exclusion mechanism used by two chemosensory systems

Luca Capello,1,* Daniele Roppolo,1,* Ve´ronique Pauli Jungo,1Paul Feinstein2andIvan Rodriguez1

1Department of Zoology and Animal Biology, and NCCR ‘Frontiers in Genetics’, University of Geneva, 30 Quai Ernest Ansermet, 1204 Geneva, Switzerland

2Department of Biological Sciences, Hunter College, CUNY, New York, USA

Keywords: gene regulation, monogenic expression, mouse, olfaction, pheromone receptor

Abstract

Sensory coding strategies within vertebrates involve the expression of a limited number of receptor types per sensory cell. In mice, each vomeronasal sensory neuron transcribes monoallelically a single V1R pheromone receptor gene, chosen from a large V1R repertoire. The nature of the signals leading to this strict receptor expression is unknown, but is apparently based on a negative feedback mechanism initiated by the transcription of the first randomly chosen functional V1R gene. We show,in vivo, that the genetic replacement of theV1rb2pheromone receptor coding sequence by an unrelated one from the odorant receptor geneM71 maintains gene exclusion. The expression of this exogenous odorant receptor in vomeronasal neurons does not trigger the transcription of odorant receptor-associated signalling molecules. These results strongly suggest that despite the different odorant and vomeronasal receptor expression sites, function and transduction cascades, a common mechanism is used by these chemoreceptors to regulate their transcription.

Introduction

In order to survive and reproduce, mammals use tools to extract

In order to survive and reproduce, mammals use tools to extract

Dans le document Vomeronasal receptors: from monogenic expression to axon guidance (Page 97-0)