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Research Report

Spatio-temporal expression pattern of receptors formyelin-associated inhibitors in the developing ratolfactory system

Marion Richard⁎, Joëlle Sacquet, François Jourdan, Véronique Pellier-Monnin1

Laboratoire Neurosciences Sensorielles, Comportement, Cognition, CNRS-UMR 5020, Université de Lyon, Lyon 1, F-69366, FranceInstitut Fédératif des Neurosciences de Lyon, Groupement Hospitalier Est, 69677 Bron Cedex, France

A R T I C L E I N F O

⁎ Corresponding author.Department of Neuro06520-8082, USA. Fax: +1 203 737 2159.

E-mail address: marion.richard@yale.edu (Abbreviations: E1, Embryonic day 1; GAP43,

Receptor; OB, Olfactory Bulb; OE, Olfactory Ep1 Present address: INSERM U842; Université

0006-8993/$ – see front matter © 2008 Elsevidoi:10.1016/j.brainres.2008.11.049

A B S T R A C T

Article history:Accepted 10 November 2008Available online 27 November 2008

The myelin-associated inhibitory proteins (Nogo-A, MAG and OMgp) that prevent axonregeneration in adult CNS, mediate their effects via a receptor referred as NgR1. Beside theirinhibitory role in the adult CNS, Nogo-A and NgR1might also be functionally involved in thedeveloping nervous system. At the present time, no detailed study is available regardingeither the onset of NgR1 expression during development or its spatio-temporal pattern ofexpression relative to the presence of Nogo-A. Two homologs of NgR1, NgR2 and NgR3, havebeen recently identified, but their function in the nervous system is still unknown in adultas well as during development. We have examined the spatio-temporal expression patternof both NgR1, NgR2 and NgR3 mRNAs and corresponding proteins in the developing ratolfactory system using in situ hybridization and immunohistochemistry. From E15–E16onwards, NgR1 mRNA was expressed by differentiating neurons in both the olfactoryepithelium and the olfactory bulb. At all developmental stages, including adult animals,NgR1 protein was preferentially targeted to olfactory axons emerging from the olfactoryepithelium. Using double-immunostainings in the postnatal olfactory mucosa, we confirmthe neuronal localization of NgR1 and its preferential distribution along the olfactory axons.The NgR2 and NgR3 transcripts and their proteins display similar expression profiles in theolfactory system. Together, our data suggest that, in non-pathological conditions, NgR1 andits homologs may play a role in axon outgrowth in the rat olfactory system and may berelevant for the confinement of neural projections within the developing olfactory bulb.

© 2008 Elsevier B.V. All rights reserved.

Keywords:Axon growthNgR1–3 receptorsSensory systemNogo-A

1. Introduction

Nogo-A, MAG (myelin-associated glycoprotein) and OMgp(oligodendrocyte-myelin glycoprotein) are myelin-associated

surgery, Yale University S

M. Richard).Growth-Associated Proteithelium; OMP, Olfactory Mde Lyon, Lyon 1, Faculté

er B.V. All rights reserved

inhibitory proteins that prevent axon regeneration in thelesioned central nervous system (CNS) of adult animals (Filbin,2003; Schwab, 2004; Liu et al., 2006; Xie and Zheng, 2008).Although structurally dissimilar, these proteins mediate their

chool of Medicine, PO Box 208082, 333 Cedar Street, New Haven, CT

in of 43 kDa; MAP2, Microtubule-Associated Protein 2; NgR, Nogo-66arker Protein; ORN, Olfactory Receptor Neuron; P1, Postnatal day 1de Médecine Laennec, UMR-S842, Lyon, F-69372, France.

.

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effects via a common neuronal receptor, the Nogo-66 receptoralso referred as NgR1 (Fournier et al., 2001; Domeniconi et al.,2002; Liu et al., 2002; Wang et al., 2002b). NgR1 is a member ofthe proteoglycan/leucine-rich repeat protein family and isattached to the cell surface by a C-terminal glycosyl-phos-phatidylinositol (GPI)-anchor (Fournier et al., 2002; He et al.,2003; Pignot et al., 2003; Yu et al., 2004) (see also Schwab et al.,2006 and Giger et al., 2008 for reviews). In order to transducethe growth inhibitory signals across the cell membrane, NgR1associates with several signaling partners, namely the p75neurotrophin receptor (p75NTR) or TROY receptor and LINGO-1,a transmembrane brain-specific protein (Wang et al., 2002a;Wong et al., 2002; Mi et al., 2004; Park et al., 2005; Shao et al.,2005). The binding of myelin-associated inhibitory proteins tothe tripartite receptor complex induces the activation of theRho GTPases pathway, resulting in the rearrangement of theactin cytoskeleton and thus leading to growth cone collapse(Niederost et al., 2002) (see also Yiu and He, 2003 for review).

In themature brain, NgR1mRNA expression is restricted tospecific sets of neurons (Hunt et al., 2002b; Josephson et al.,2002; Mingorance et al., 2004; Hasegawa et al., 2005; Barrette etal., 2007; Funahashi et al., 2008). NgR1 protein is mainlydetected in neuronal cell bodies and to a lesser extent inneuritic processes (Wang et al., 2002c). There is a generalagreement that Nogo-A/NgR1 interaction constitutes one ofthe major impediments to axonal regeneration in the injuredCNS (Filbin, 2003; Schwab, 2004; Liu et al., 2006; Xie and Zheng,2008). Nevertheless, several data suggest that Nogo-A/NgR1may also have a physiological function in the intact adult CNS,unrelated to injury or regeneration. Notably, it has been shownthat Nogo-A/NgR1 interaction may stabilize mature myeli-nated fiber tracts by preventing unnecessary axonal sprouting,thus contributing to the maintenance of specific connections(Wang et al., 2002c; Raisman, 2004; McGee et al., 2005).

Beside these functions in the adult CNS, it becomes moreandmore obvious that Nogo-A andNgR1might also play a rolein the developing nervous system. NgR1 mRNA is expressedduring the nervous system development from late embryonicstages in fish, rodent and human (Hunt et al., 2002b; Josephsonet al., 2002; Klinger et al., 2004). Both Nogo-A and NgR1transcripts have been detected in neocortical neurons, inhippocampal pyramidal neurons and in motor neurons of thespinal cord in several species (Josephson et al., 2001; Joseph-son et al., 2002; Mingorance et al., 2004). By contrast, littleinformation is available regarding the expression patterns ofNogo-A and NgR1 proteins in the developing nervous system.Several studies have shown that Nogo-A protein is present insome neuronal populations during brain development, with apreferential distribution within outgrowing axons (Huber etal., 2002; Tozaki et al., 2002; Wang et al., 2002c; Mingorance etal., 2004; Mingorance-Le Meur et al., 2007). However, nodetailed study is yet available about the onset of NgR1expression in the developing nervous system and its spatio-temporal distribution relative to the presence of Nogo-A. Werecently reported that Nogo-A is detected within outgrowingolfactory axons, at both developmental and adult stages,supporting a role of this protein in axon outgrowth in theintact nervous system (Richard et al., 2005). In order todetermine whether the NgR1 receptor might be involved inNogo-A signaling in the developing olfactory system, we study

here the spatio-temporal pattern of expression of both NgR1mRNA and protein in this system. We also describe theexpression profiles of two newly identified members of theNgR family, NgR2 and NgR3, that share common structuralfeatures with NgR1 (Barton et al., 2003; Lauren et al., 2003;Pignot et al., 2003). Our results show that NgR1, NgR2 andNgR3mRNAs and proteins display similar neuronal expressionpatterns in the developing olfactory system, with a prefer-ential targeting of NgR proteins to olfactory axons.

2. Results

2.1. Spatio-temporal profile of NgR1 expression in thedeveloping olfactory system

2.1.1. In situ hybridization dataThe first NgR1 mRNA expression in the olfactory system wasdetected in E16 embryos. NgR1 mRNA appeared weaklyexpressed by some differentiating cell bodies located in theolfactory epithelium and in the marginal zone of the devel-oping olfactory bulb (Fig. 1A). Note that no signal was detectedin the dividing progenitor cells from the ventricular zone. InE16 embryos as in later embryonic stages, no signal wasobserved in adjacent sections hybridized with the sense probe(Fig. 1B). From E18 (Fig. 1C) to E21 (Fig. 1D), numerous NgR1mRNA-positive cells were present in both the olfactory andvomeronasal epithelia. As shown in Fig. 1G, a strong accumu-lation of NgR1 transcript was also detected in the presumptivemitral cell layer of the olfactory bulb in E21 embryos.

In young postnatal rats (P1–P6), a sustained NgR1 mRNAexpression was observed in most neuronal cell bodiesdistributed throughout the thickness of the olfactory andvomeronasal epithelia (Fig. 1E). In the olfactory bulb, a strongstaining for NgR1 mRNA was displayed by the mitral cellbodies (Fig. 1H). In addition, a faint signal was also detected atP6 in periglomerular or tufted cells located on the internal sideof the glomeruli (Fig. 1H).

In P15 and P40 rats, a weak to moderate NgR1 mRNAexpression was observed in cell bodies widely distributed inthe whole thickness of the epithelium (Fig. 1F). Consideringthe number and the distribution of the labeled cell bodieswithin the epithelium, we can assume that most olfactoryreceptor neurons (ORNs) express NgR1 mRNA, includingmature ORNs located in the middle part of the epithelium(Farbman andMargolis, 1980; Schwob, 2002). No specific signalwas detected in the adjacent sections hybridized with thesense probe, thus confirming the specificity of the labeling(Fig. 1F'). In the adult olfactory bulb, the pattern of NgR1mRNAexpression was similar to that reported in younger rats,although the hybridization signal seemed less intense (Fig. 1I).

2.1.2. Immunohistochemical dataThe immunohistochemical analysis was carried out onsections of the olfactory epithelium and olfactory bulb byusing two different polyclonal anti-NgR1 antisera. Sincesimilar resultswere obtainedwith both antibodies, we decidedto present only data collected with the anti-NgR1 antibody,which has been previously characterized and validated(Venkatesh et al., 2005).

Fig. 1 – NgR1mRNA expression in the developing rat olfactory system (in situ hybridization on coronal sections). At E16 (A), a weakexpression of NgR1mRNA is detected in the developing olfactory epithelium (OE, arrowheads). Themarginal zone (MZ, arrows)of the presumptive olfactory bulb exhibits a moderate labeling compared to the ventricular zone (VZ). No signal is observed in thedeveloping olfactory epithelium as well as in the presumptive olfactory bulb with the sense probe (B). An increased number ofstained ORNs is found in the olfactory epithelium at E18 (C) and in the vomeronasal organ (VNO, arrowheads) at E21 (D). At P6 (E),most neuronal cell bodies appear strongly labeled in the vomeronasal epithelium (arrowheads). At P40 (F), NgR1mRNA remainsexpressed byORNsdistributed throughout the epithelial thicknesswhile the sense probe gives no specific signal (F'). At E21 (G), inthe developing olfactory bulb, a sustained staining is observed in the future mitral cell layer (Mi, arrows). At P6 (H), inaddition to labeled mitral cells (arrows), a positive signal is detected in tufted or periglomerular cells located on the internalside of glomeruli (Gl, arrowheads). In the adult olfactory bulb (I), NgR1 mRNA remains expressed by the mitral (arrows)and tufted or periglomerular (arrowheads) cells but with a decreased intensity compared with earlier postnatal stages.EPL: External Plexiform Layer; Gr: Granule cell layer; LP: Lamina Propria; ONL: Olfactory Nerve Layer; ORNs: OlfactoryReceptor Neurons; pGl: presumptive Glomerular layer; pONL: presumptive Olfactory Nerve Layer. Scale bars=100μm (A–B, D–E, G–I), 50 μm (C, F and F').

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Fig. 2 – NgR1 protein expression in the developing rat olfactory system (immunostaining on coronal sections). At E16 (A),olfactory axons emerging from the olfactory epithelium (OE, arrows) and grouping together into the presumptive olfactorynerve layer (pONL) are well NgR1-immunoreactive. Note the presence of a few weakly labeled neuronal cell bodies in thesensory epithelium (arrowheads). At E18 (B) and E21 (C), NgR1 is still detected in axon fascicles extending from the olfactory(arrows in B) and vomeronasal (arrows in C) epithelia. No specific staining is observed on control sections (see inset in B).At P1 (D), P15 (E) and P40 (F), arrows point to strongly NgR1-immunoreactive olfactory axon fascicles (Ax) coursing through thelamina propria (LP). Thick NgR1-immunopositive vomeronasal axon fascicles are indicated by arrowheads in D, E and F. Inthe neonate olfactory bulb (G), NgR1-immunoreactivity is detected in the olfactory nerve layer (ONL) and the presumptiveglomerular layer (pGl). At P6 (H), as indicated by arrows, labeled olfactory glomeruli are visible in the glomerular layer (Gl). In P40olfactory bulb (I), the olfactory nerve layer appears NgR1-immunopositive while no more evident staining is observed in theglomeruli. Note the weak labeling of mitral (Mi, arrows) and periglomerular or tufted (arrowheads) cell bodies. EPL: ExternalPlexiform Layer; Gr: Granule cell layer; IPL: Internal Plexiform Layer; MZ: Marginal Zone; VNO: Vomeronasal Organ. Scalebars=50 μm (A, E), 100 μm (B and inset, C–D, F–I).

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The first NgR1 protein expression in the developingolfactory system was detected in E15–E16 embryos. At thesestages, an obvious NgR1-labeling was noted in axon fasciclesemerging from the olfactory epithelium and the vomeronasalorgan and extending towards the olfactory bulb (Fig. 2A). FromE18 onwards, a sustained immunoreactivity was associatedwith olfactory and vomeronasal axon bundles (Figs. 2B and C).At all embryonic stages, only fewweakly stained neuronal cellbodies were observed in the thickness of both sensoryepithelia. No specific labeling was detected in control sectionsinwhich the primary antibodywas omitted (inset in Fig. 2B). Innewborn rats, olfactory and vomeronasal axons still exhibiteda marked NgR1 expression that contrasted with the weakNgR1-staining of neuronal cell bodies in both olfactory andvomeronasal epithelia (Fig. 2D). This pattern of labelingpersisted in P15 (Fig. 2E) and adult animals (Fig. 2F).

From E15 to E18, the olfactory bulb displayed a moderateNgR1-labeling in the olfactory nerve layer (Figs. 2A and B)while the other layers appeared weakly and diffusely stained.From E21 onwards, the expression pattern of NgR1 proteinbecame restricted to discrete bulbar layers. The mostprominent NgR1-immunoreactivity was observed in theolfactory nerve layer and the presumptive glomerular layer(Figs. 2G and H). At P1, and more clearly at P6, the newlyformed olfactory glomeruli exhibited an obvious NgR1-immunostaining (Figs. 2G and H). Nevertheless, this glomer-ular labeling appeared transitory since NgR1-immunoreac-tivity was no longer observed in olfactory glomeruli at P15and P40 (Fig. 2I). In adult rats, the olfactory bulb displayed aweak staining in the olfactory nerve layer and numerousfaintly immunolabeled cell bodies in the periglomerular andmitral cell layers (Fig. 2I). Whatever the developmental stagestudied, the NgR1 protein was never detected in the axon anddendrites of mitral cells.

2.1.3. Neuronal localization of NgR1 in the olfactory systemIn order to confirm the neuronal localization of NgR1 in thedeveloping olfactory mucosa, double-labeling experimentswere carried out by combining the anti-NgR1 antibody withtwo ORN markers, GAP43 (Growth-associated Protein of43 kDa) and OMP (Olfactory Marker Protein). These experi-ments were performed on tissue sections from P6 rats, at astage where NgR1 protein was easily detected within olfactoryaxon bundles. At P6, the population of olfactory receptorneurons is mainly composed of differentiating neuronscharacterized by the expression of GAP43 (Verhaagen et al.,1989; Verhaagen et al., 1990; Schwob, 2002). NgR1- and GAP43-immunoreactivities showed a similar distribution in olfactory

Fig. 3 – Neuronal expression of NgR1 protein in the olfactory sysGAP43-stained olfactory axons (Ax, arrows in B), extending from tpropria (LP), are strongly NgR1-immunoreactive (arrows in A). Ax(arrows in C). (D–F) NgR1 protein (D) colocalizes with OMP (E) in omerged image (arrows in F). (G–I) Thin S100β-immunopositive prodisplay any staining for NgR1 (G), but are closely apposed to NgROMP-positive olfactory axon terminals (K) are also NgR1-immunvisible on the merged image (L). (M–O) In the developing glomeruMAP2-immunoreactive dendritic processes (N). Both stainings diEPL: External Plexiform Layer; ONL: Olfactory Nerve Layer. Scale

axon bundles coursing through the lamina propria (Figs. 3Aand B). At this postnatal stage, some olfactory receptorneurons express the OMP protein, a selective marker of fullydifferentiated ORNs whose axons have reached their target(Keller and Margolis, 1975; Farbman and Margolis, 1980;Schwob, 2002). OMP-immunopositive olfactory axon fascicles,located in the lamina propria, were also immunoreactive forNgR1 (Figs. 3D and E). The colocalization of NgR1 protein withOMP or GAP43 in olfactory axons was clearly demonstrated byan obvious overlapping on the merged images (Figs. 3C and F),confirming the neuronal expression of NgR1 by both imma-ture and mature ORNs.

Because the olfactory axon fascicles are closely associatedwith olfactory ensheathing cell processes in the laminapropria, we tested the possibility that NgR1 protein may bejointly expressed by these glial cells. To do so, tissue sectionswere double-stained for NgR1 and S100β, a glial markerspecifically expressed by olfactory ensheathing cells (Astic etal., 1998; Au et al., 2002). As shown in Figs. 3G–I, thin stronglyS100β-positive glial processes enwrapped NgR1-immunoreac-tive olfactory axons, but the staining patterns of each markerwere adjacent and non-overlapping, attesting that NgR1expression was exclusively neuronal.

Otherwise, to confirm that NgR1 is restricted to olfactoryaxon terminals within the glomerular layer, we performeddouble-labeling experiments combining the anti-NgR1 anti-body with antibodies directed against OMP, an axonal marker,or MAP2 (Microtubule-Associated Protein 2), a marker thatspecifically labels dendrites extending into both the externalplexiform and glomerular layers of the olfactory bulb (Bailey etal., 1999; Kasowski et al., 1999; Treloar et al., 1999). As shown inFigs. 3J–L, there was an evident overlap between NgR1 andOMP stainings in the glomeruli, confirming the localization ofNgR1 in olfactory axon terminals. In contrast, NgR1- andMAP2-immunostainings never colocalized in the olfactoryglomeruli: MAP2-positive dendritic processes were clearlydistinct from NgR1-positive axon terminals (Figs. 3M–O).

2.2. Expression profiles of NgR2 and NgR3 in thedeveloping olfactory system

2.2.1. In situ hybridization dataThe distribution of NgR2 and NgR3 transcripts in the develop-ing olfactory system was similar to that described above forNgR1. The first expression of NgR2 mRNA was detected in theolfactory and vomeronasal epithelia of E16 embryos. At E18,several neuronal cell bodies were stained by the NgR2antisense probe in the olfactory epithelium (Fig. 4A), while

tem at P6 (confocal analysis on coronal sections). (A–C)he olfactory epithelium (OE) and coursing through the laminaonal colocalization is clearly visible on the merged imagelfactory axon bundles. Overlapping is detected on thecesses of olfactory ensheathing cells (arrowheads in H) do not1-immunoreactive axons on the merged image (I). (J–L)oreactive in the developing glomerulus (Gl). Colocalization islus, NgR1-positive axons (M) are clearly distinct fromsplay obviously no colocalization on the merged image (O).bars=20 μm (A–F, J–O), 10 μm (G–I).

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hybridization of the adjacent section with a sense proberesulted in background level of staining (Fig. 4A'). A gradualincrease in the number of labeled neurons was observed withage in both sensory epithelia so thatmost neuronal cell bodiesexhibited a sustained NgR3mRNA signal at P6 (Figs. 4B and C).In adult olfactory epithelium, NgR3 mRNA expression wasdetected in cell bodies located in the middle compartment ofthe epithelial thickness, just above the basal cells (Figs. 4D).This staining pattern clearly shows that this transcript isexpressed by mature ORNs.

In the developing olfactory bulb, the distribution patternsof NgR2 and NgR3 mRNAs were similar to that reported forNgR1. From P1 onwards, the expression of both transcriptswas detected in the mitral cell layer and then extended toperiglomerular or tufted cells located on the internal side ofthe glomeruli in P6 and P40 olfactory bulbs (Fig. 4E).

2.2.2. Immunohistochemical dataThe immunohistochemical study of NgR2 and NgR3 wasperformed by using antisera known to allow the specificdetection of these NgR family members in the adult brain(Venkatesh et al., 2005). The spatio-temporal patterns of NgR2and NgR3 proteins expression were similar to that describedfor NgR1 in the developing olfactory system. At E16, axonalprocesses extending from the olfactory epithelium appearedwell-labeled along their course in the lamina propria, whereasneuronal cell bodies displayed a weak immunoreactivity (Fig.4F). As shown at E21, vomeronasal axons are also NgR2 andNgR3-immunoreactive (Fig. 4G). Marked NgR2- and NgR3-stainings of olfactory axon bundles were still observed in P15and adult rats (Fig. 4H). No labeling was detected in theolfactory system when the anti-NgR2 or anti-NgR3 antibodieswere omitted (data not shown).

At early embryonic stages (E14–E16), NgR2 and NgR3proteins were predominantly detected, as for NgR1, in thepresumptive olfactory nerve layer of the developing olfactorybulb (Fig. 4F). At P1 and P6, in addition to the well-labeledolfactory nerve layer, discrete NgR2- and NgR3-stainingsappeared in the presumptive glomerular layer (Fig. 4I). Aspreviously reported for NgR1, the labeling of olfactoryglomeruli was transient and not further detected in P15 andadult rats (Fig. 4J). In the adult olfactory bulb, moderate NgR2-and NgR3-immunoreactivities persisted within the olfactorynerve layer, whereas periglomerular and mitral cell bodiesappeared weakly stained with the two antibodies (Fig. 4J).

3. Discussion

The present study describes for the first time, the develop-mental expression of the Nogo-66 receptor, NgR1 and of itshomologs, NgR2 and NgR3, in the rat olfactory system, bycombining in situ hybridization and immunohistochemistry.The main results of our study are summarized in Table 1.

3.1. Developmental expression of NgR1 and its homologsin the olfactory system

During development, the number of olfactory receptor neu-rons expressing NgR1 mRNA rapidly increases with age from

E16 onwards and is maintained in the adult olfactoryepithelium. Mitral cells also display a sustained stainingfor NgR1 mRNA starting at E18, confirming previous reportsat embryonic, postnatal and adult stages (Josephson et al.,2002; Lauren et al., 2003; Hasegawa et al., 2005). Theexpression of NgR1 mRNA is thus concomitant with theperiod of intense neurogenesis and neuronal differentiationthat extends over embryonic and postnatal stages in the ratolfactory system (Verhaagen et al., 1989; Verhaagen et al.,1990; Pellier et al., 1994; Schwob, 2002). Notably, thesequential expression in the olfactory bulb neurons corre-lates with the chronology of their differentiation, sincemitral cells begin differentiating in the late embryonicstages, while tufted and periglomerular cells appear lateron and mature mostly after birth (Mair et al., 1982; Brunjesand Frazier, 1986; Blanchart et al., 2006). Our results areconsistent with previous studies reporting high levels ofNgR1 mRNA expression in mature neurons of the adultnervous system such as the cerebral cortex, the hippocam-pus, the thalamus and the cerebellum in rodent and humanspecies (Fournier et al., 2001; Hunt et al., 2002b; Josephson etal., 2002; Josephson et al., 2003; Hasegawa et al., 2005; Zhenget al., 2005; Barrette et al., 2007; Funahashi et al., 2008) (seeHunt et al., 2002a for review). Our data also add the olfactorysystem to the several areas of the developing nervoussystem expressing NgR1 mRNA and thereby further supportthe hypothesis of a function of NgR1 during development(Josephson et al., 2002; Josephson et al., 2003; Lauren et al.,2003; Mingorance et al., 2004; O'Neill et al., 2004; Al Halabiahet al., 2005).

Consistent with the onset of NgR1 mRNA expression, wedemonstrate the presence of the NgR1 protein in growingolfactory axons from embryonic stages E15–E16 and a rapidincrease of the number of NgR1-immunolabeled fascicleswith age. Whatever the age and the state of ORN differentia-tion (immature versus mature), the NgR1 protein is prefer-entially targeted to the axonal compartment, since itcolocalizes with GAP43 and OMP, two ORN axonal markers(Verhaagen et al., 1989; Pellier et al., 1994). The partialcolocalization might reflect a different subcellular distribu-tion of the cytoplasmic GAP43 or OMP protein and theplasma membrane receptor NgR1 (Fournier et al., 2001). Inaccordance with this hypothesis, NgR1 and PSA-NCAM, anaxonal plasma membrane marker (Aoki et al., 1999), display ahigher degree of colocalization (data not shown). Thetargeting of NgR1 protein to outgrowing axonal processeswas already reported in chick embryonic spinal cord explants(Fournier et al., 2001) and in rat postnatal cerebellar neuronsin vitro (Domeniconi et al., 2002) as well as more recently inthe mouse retinal ganglion cells both in vivo and in vitro(Wang et al., 2008). NgR1 was also detected in myelinatedaxonal profiles in the adult cortex and spinal cord of rodentsin vivo (Wang et al., 2002c). Interestingly, in the olfactorybulb, NgR1 protein was restricted to mitral and tufted orperiglomerular cell bodies, without any labeling of dendriticand axonal processes, showing that targeting of NgR1 toneurites may be dependent on the cell type or the expressionlevel.

Interestingly, double-labeling experiments combiningNgR1 and the S100β glial marker, indicate that the olfactory

Fig. 4 – NgR2 and NgR3 mRNAs and proteins expression in the developing rat olfactory system (in situ hybridization andimmunohistochemistry on coronal sections). (A–E) Expression of NgR2 and NgR3 mRNAs. At E18 (A), numerous neuronalcell bodies express NgR2 transcript in the developing olfactory epithelium (OE, arrowheads), while no specific signal isdetected with the sense probe (A'). At P6, an increasing number of NgR3 mRNA expressing neurons is found in the olfactoryepithelium (arrowheads in B) and in the vomeronasal organ (VNO, arrowheads in C). In the adult olfactory epithelium (D),NgR3mRNA expression is detected in thewholemiddle compartment of the epithelial thickness (inset in D). In P6 olfactory bulb(E), the mitral cells strongly express NgR2 mRNA (Mi, arrows in E) and a moderate signal is observed in the periglomerularor tufted cells, that are located on the internal side of the glomeruli (Gl, arrowheads in E). (F–J) Expression of NgR2 and NgR3proteins. At embryonic stage E16 (F) NgR2 protein is detected in olfactory axons (arrows in F) aggregating into the presumptiveolfactory nerve layer (pONL). At E21, vomeronasal axon bundles are also NgR2-positive (Ax, arrow in G). Immunoreactivityfor NgR2 protein is still observed in olfactory axon bundles at P15 (arrow in H). At P6 (I), an evident NgR2-staining isdetected in the olfactory nerve layer (ONL) and a moderate labeling is also observed in the developing glomerular layer (Gl,arrows in I). In P40 olfactory bulb (J), NgR3-immunoreactivity is present in the olfactory nerve layer and more weakly in mitral(Mi, arrows) and periglomerular or tufted cell bodies (arrowheads). EPL: External Plexiform Layer; Gr: Granule cell layer;IPL: Internal Plexiform Layer; LP: Lamina Propria; MZ: Marginal Zone; pGl: presumptive Glomerular layer. Scale bars=50 μm (A,A', B and inset in D), 100 μm (C–J).

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ensheathing cells do not express NgR1 protein in vivo. This isslightly different from studies reporting the presence of NgR1protein in restricted glial populations of the nervous system invivo (Liao et al., 2004; Liu et al., 2005; Satoh et al., 2005) andboth NgR1mRNA and protein in olfactory ensheathing cells invitro (Woodhall et al., 2003; Su et al., 2007). This discrepancy isvery likely explained by the well-described variations of thephenotype of olfactory ensheathing cells, depending on the invitro or in vivo conditions (Devon and Doucette, 1992;Franceschini and Barnett, 1996; Alexander et al., 2002). Inconclusion, NgR1 displays an exclusive neuronal expressionand an axonal distribution in the peripheral olfactory systemthroughout development.

In addition, the present study demonstrates that NgR2and NgR3 mRNAs and proteins are concomitantly expressedin the ORNs and bulbar neurons during development and inadult, with a cellular distribution similar to NgR1. Previousstudies have reported widely overlapping expressions of thethree NgR transcripts but only in the adult nervous system(Lauren et al., 2003; Pignot et al., 2003; Barrette et al., 2007;Funahashi et al., 2008). Our results expand this observationto the olfactory system both during development and inadult. Fewer results were available about the expression ofNgR2 and NgR3 proteins. Western blot analyses havereported their presence in the adult nervous system buttheir neuronal distribution was fairly described (Pignot et al.,

Table 1 – Expression of NgR1 mRNA and protein in thedeveloping olfactory system. NgR2 and NgR3 mRNAs andthe corresponding proteins display similar expressionpatterns

E15–E18 P1–P6 P15–P40

NgR1 mRNAVomeronasal neurons + ++ ++ORNs + ++ ++Mi + ++ +PG or tufted cells n.i +/− +Gr − − −

NgR1 proteinVomeronasal axons + ++ ++Olfactory axons + ++ ++ONL + ++ +Gl n.i + −EPL n.i − −Mi +/− +/− +/−IPL n.i − −Gr − − −

‘++’=strong staining, ‘+’=moderate staining, ‘+/−’=weak staining,‘−’=no staining, ‘n.i’=cellular population or bulbar layer not yetidentifiable at this developmental stage.EPL: External Plexiform Layer; Gl: Glomerular layer; Gr: Granulecell layer; IPL: Internal Plexiform Layer; Mi: Mitral cells; ONL:Olfactory Nerve Layer; ORNs: Olfactory Receptor Neurons; PG:Periglomerular cells.

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2003; Venkatesh et al., 2005). NgR3 (NgRH2 in their study) ispresent in adult rat hippocampal, cortical, cerebellar andthalamic neurons (Pignot et al., 2003). NgR2 is detected inaxons of the adult optic nerve and in axonal profiles ofcultured neonatal DRG neurons expressing ectopically thishomolog (Venkatesh et al., 2005). Our study thus demon-strates that in in vivo conditions, the three members of theNgR family are localized in olfactory axons during develop-ment as well as in adult.

3.2. Relation of NgRs and Nogo-A expression patterns

3.2.1. In the peripheral olfactory systemAmong the myelin-associated inhibitory proteins, Nogo-A isknown as one of the ligands interacting with NgR1. In aprevious study, we demonstrated that the Nogo-A transcriptand protein are strictly restricted to immature ORNs duringdevelopment and in adult (Richard et al., 2005). Since NgR1receptor is detected in most ORNs, whatever their level ofdifferentiation, the coexpression of the ligand and its receptorcould only be seen in the population of differentiating ORNs.Furthermore, we show in the present study that NgR1 proteinis preferentially targeted to olfactory axon processes, aspreviously observed for Nogo-A (Richard et al., 2005). Thecoexpression of NgR1 and Nogo-A in immature ORNs and thepreferential distribution of both proteins in olfactory axonssupport the assumption that theymight interact at the level ofaxon bundles during development. Indeed, Nogo-A is exposedto the membrane surface of cultured neuronal cells where itcould interact locally with NgR1 (Dodd et al., 2005; Mingor-ance-Le Meur et al., 2007). However, this hypothesis seems

unlikely in the model of the olfactory system since ourprevious results rather argue for an intracellular distributionof Nogo-A in olfactory axons (Richard et al., 2005). Thisintracellular localization of Nogo-A is also supported by invitro experiments in which Nogo-A-immunoreactivity wasundetectable in outgrowing olfactory axons when cell per-meabilization was omitted (unpublished personal observa-tions). Finally, the p75NTR and TROY co-receptors which arerequired for Nogo-A/NgR1 signal transduction, are neverexpressed by ORNs, but are restricted to olfactory ensheathingcells (Gong et al., 1994; Hisaoka et al., 2004).

The hypothesis that NgR1 andNogo-Amight not interact inthe peripheral olfactory system is reinforced by some dis-tinctive features of their respective expression patterns. Wehave established that NgR1 expression begins in the ORNs atE15–E16 and is delayed compared to Nogo-A which is detectedin the pioneer outgrowing olfactory axons as early as E13(Richard et al., 2005). Such a lack of temporal correlationbetween the onset of Nogo-A and NgR1 expressions was alsoreported during embryonic development in the human spinalcord (O'Neill et al., 2004) as well as in the mouse hippocampusand optic pathway (Mingorance et al., 2004; Wang et al., 2008).Moreover, NgR1 expression is not restricted to axons incontact with myelin containing Nogo-A, since NgR1 isdetected in small unmyelinated axons in the adult brain(Wang et al., 2002c) and in the peripheral olfactory system(this study). Interestingly, Lingo-1, a component of the Nogoreceptor complex, is also expressed by neurons lackingmyelinsheaths (Llorens et al., 2008). All these observations suggestthat neuronal expression of the Nogo receptor complex canoccur independently of the presence of myelin-associatedinhibitory proteins, raising the possibility that NgR1 andNogo-A might exert proper independent functions that do notinvolve the classically described ligand-receptor interaction.

An attractive hypothesis may be that NgR1 mediatesreceptor–receptor interactions at the plasma membranesurface of olfactory axons. In support to this hypothesis,NgR1 leucine-rich repeats (LRRs) enable the formation ofhomodimers (Fournier et al., 2002; Liu et al., 2002) and mayprovide a structural framework for additional protein–proteininteractions (Kobe and Kajava, 2001; Chen and Liu, 2005).Regarding NgR2 and NgR3, it should be noted that MAG is theonly known ligand for NgR2 and that no ligand has been yetidentified for NgR3 (Venkatesh et al., 2005; Lauren et al.,2007). Whether MAG may interact with NgR2 at the surface ofolfactory axons is still unknown, since MAG expression hasnot been studied in the developing olfactory system. BecauseNgR2 and NgR3 receptors also possess LRR domains (Bartonet al., 2003; Lauren et al., 2003; Pignot et al., 2003), they mightbe involved in homo- or heterophilic bindings with thedifferent NgR homologs. Such interactions might modulatethe adhesive properties of olfactory axons and thereby play arole in axon outgrowth occurring during development and inadult. The concept, according to which receptors can beinvolved in axon growth independently of binding to theirspecific ligands, was demonstrated for the Robo receptors.Indeed, these receptors form hetero- and homophilic inter-actions and thereby modulate the in vitro axon outgrowth ofolfactory bulb neurons in the absence of their ligands Slit(s)(Hivert et al., 2002).

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3.2.2. In the olfactory bulbA transient expression of the NgR family members isobserved in the developing glomerular layer between P1and P6, whereas no labeling is further detected in olderanimals. The presence of NgR1 and its homologs withinglomeruli coincides with the formation of glomeruli thatmostly occurs during the first postnatal week in rodents(Meisami and Sendera, 1993; Bailey et al., 1999; Treloar et al.,1999). This postnatal period is characterized by a remarkablerearrangement of olfactory axon terminals and dendriticarbors, allowing the establishment and maturation of func-tional synapses within glomeruli (Valverde et al., 1992; Akinsand Greer, 2006; Blanchart et al., 2006; Walz et al., 2006;Blanchart et al., 2008). Based on our NgR1/OMP and NgR1/MAP2 double-immunostaining experiments, we demonstratethat NgR1 glomerular labeling is exclusively associated witholfactory axon terminals and not with the dendrites of bulbarneurons. Interestingly, we previously reported a transientexpression of Nogo-A within the dendritic processes of mitralcells at developmental stages which coincide with thepresence of significant amounts of NgR1 in olfactory axonterminals (Richard et al., 2005). The concomitant presence ofNgR1 and Nogo-A raises the possibility that they mightcontribute to axodendritic interactions occurring during theformation of glomeruli. Dendritic Nogo-A might exert a localinhibitory effect inducing the arrest of olfactory axon growthwithin the glomerular layer and thereby contribute to therestriction of exuberant axon growth reported in theolfactory bulb during the early postnatal period (Klenoffand Greer, 1998). Other studies also support the hypothesisthat Nogo-A/NgR1 interaction may restrict fiber growth andterminal arborization within a target area in the postnatalnervous system. For instance, interfering with Nogo-A/NgR1signaling during development induces an aberrant axonalsprouting in the optic nerve (Colello et al., 1994) and thecorticospinal tract (Schwab and Schnell, 1991). Similarobservations have also been reported in adult animals forthe intact Purkinje cell axons (Buffo et al., 2000) and thesensory-motor corticospinal fibers (Bareyre et al., 2002).

In conclusion, our data favor the hypothesis that Nogo-Aand NgRs play a role in axon outgrowth and contribute to theconfinement of neural projections within the developingolfactory bulb. However, additional experiments are requiredto determine if such a developmental function of NgR familymembers is related or not to the presence of Nogo-A and/orother unidentified ligands. Several recent papers clearlyshowed that NgR1 has multiple additional ligands, includingRTN3, another member of the reticulon family (Lauren et al.,2007), the amyloid precursor protein (Park et al., 2006a,b) andFibroblast Growth Factor 1–2 (Lee et al., 2008). Because theseproteins are expressed by the outgrowing olfactory axons orthe olfactory ensheathing cells (Clarris et al., 1995; Mackay-Sim and Chuah, 2000; Kumamaru et al., 2004), the possibilityexists but needs further investigation that they may generatenew functional interactions with NgR1 in the developingolfactory system. Since NgR1 has been recently involved in agrowing number and variety of processes, notably neuronalcell death (Dupuis et al., 2008), synaptic plasticity (Lee et al.,2008), neuroinflammation and diseases (David et al., 2008), itappears particularly relevant to determine the subcellular

distribution and the physiological function of NgR1, as well asits homologs, in the non-injured nervous system. Our data inthe developing olfactory system emphasize an additionalregion of the nervous systemwhere those important functionsmight be exerted.

4. Experimental procedures

4.1. Animals

Wistar-SPF rat embryos from the 13th to the 20th day ofgestation (day of conception=E1) and rats aged of 1, 6, 15 and40 postnatal days (day of birth=P1) were purchased fromCharles River (St. Germain sur l'Arbresle, France). For eachdevelopmental stage, at least two different litters were used.Animals were kept in a 12-hour light/dark cycle and providedwith food and water ad libitum. The handling of animals wasconducted according to European Community Council Direc-tive (86/609/EEC) and French Ethical Committee for the careand use of laboratory animals.

4.2. Preparation of tissues

Time-pregnant dams were deeply anesthetized withEquithesin (0.5 ml/100 g body weight) and embryos weretaken out by cesarean section. Embryos were quicklydecapitated and heads were immersed in cold fixative during4 h for younger embryos and overnight for the others. For insitu hybridization, the fixative consisted in 4% paraformal-dehyde diluted in phosphate buffer 100 mM (PB). Forimmunohistochemistry, tissues were fixed with periodate–lysine–paraformaldehyde fixative, consisting of 2% parafor-maldehyde, 75 mM lysine, 10 mM sodium m-periodate in50 mM phosphate-buffered saline (PBS) (McLean and Nakane,1974).

Postnatal and adult rats were deeply anesthetized withEquithesin and transcardially perfused with a Ringer's solu-tion followed by the same fixatives. The nasal cavity and theolfactory bulbs were carefully dissected out and post-fixedovernight at 4 °C. All tissues were then cryoprotected insucrose (10% in PB 100 mM), frozen at −55 °C in nitrogen-cooled isopentane and stored at −75 °C until use. Coronalsections (14 μm-thick) were seriallymade on a Leitz cryostat at−20 °C and thaw-mounted on gelatin-coated glass slides forimmunohistochemistry (stored at −20 °C until use) or onSuperfrost© Plus glass slides (Menzel-Gläser, CML, France) forin situ hybridization (stored at −75 °C until use).

4.3. In situ hybridization

In situ hybridization against NgR1 and its homologs wasperformed using specific digoxygenin(DIG)-labeled ribo-probes. Plasmids containing the mouse NgR1 cDNA or therat NgR2 (=NgRH1) and NgR3 (=NgRH2) cDNAswere previouslydescribed (Hunt et al., 2002b; Pignot et al., 2003). DIG-labeledriboprobes were generated using a RNA labeling kit and theirconcentration was determined by dot blot, according to themanufacturer's recommendations (Roche diagnostics, Mey-lan, France). The specificity of the signal was verified by

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comparison with adjacent sections processed with senseprobes under identical conditions.

Hybridization protocol was similar to that described in arecent study (Richard et al., 2005). Briefly, the probes werediluted to a final concentration of 3–5 μg/ml in hybridizationbuffer containing 50% deionized formamide (Sigma-Aldrich),10% dextran sulfate (Bioprobes, Montreuil, France), 1% block-ing reagent (Roche diagnostics), 10 mM Tris–HCl, 300mMNaCland 1 mM EDTA. Hybridization was performed overnight at65 °C. After several washes and RNase treatment, overnightincubation was processed at 4 °C with alkaline phosphatase-conjugated anti-DIG antibody (Roche diagnostics) diluted at1:1000 in incubation buffer (0.05% Triton X-100, 1% blockingreagent, 100 mM Tris–HCl and 150 mM NaCl, pH 7.5). Sectionswere developed in the dark at room temperature withrevelation buffer containing 0.33 mg/ml 4-nitroblue tetrazo-lium chloride and 0.175 mg/ml 5-bromo-4-chloro-3-indolylphosphate (Roche diagnostics).

4.4. Antibodies

Three rabbit polyclonal antibodies directed against NgR1,NgR2 and NgR3 respectively were used in this study. Antiserahave been raised against the C-terminal “unique” domain ofthe homologs, the stalk region (Venkatesh et al., 2005). Thespecificity of each antibody for the different members of theNgR receptor family and the absence of cross-reactivity havebeen previously assessed by western blot analysis andimmunohistochemistry on transfected COS cells, as well aswestern blots on rat brain extracts (Venkatesh et al., 2005).NgR1 and NgR2 were used at the dilution 1:2000 and NgR3 atthe dilution 1:500. In addition, we used a rabbit polyclonalanti-NgR1 antibody commercially available (1:4000, cloneAB5615, Chemicon, France). Double-immunostainings wereperformed by combining the non-commercial anti-NgR1antibody with either different mouse monoclonal antibodiesrespectively directed against growth-associated protein of43 kDa (GAP43, 1:600, clone GAP-7B10, Sigma-Aldrich),microtubule-associated protein 2 (MAP2, 1:400, clone HM-2,Sigma-Aldrich) and S100β subunit (1:400, clone SHB1, Sigma-Aldrich) or a goat polyclonal antibody raised against olfactorymarker protein (OMP, 1:800, gift from Dr. F. Margolis,University of Maryland, Baltimore, MD, USA) (Keller andMargolis, 1975).

4.5. Immunohistochemistry

The specificity of each immunolabeling was assessed oncontrol sections in which the primary antibody was omitted.No specific staining was detected using this procedure for allthe developmental stages analyzed. We processed singlelabelings for each anti-NgR homolog antibody by visualizingperoxydase activity. Fluorescent double-immunostainingswere processed by coupling the anti-NgR1 antibody withneuronal or glial markers (see antibody section).

Sectionswere air-dried for 10min at room temperature andrehydrated in PBS (pH 7.4). Endogenous peroxidases werequenched by 30 min incubation in PBS-H2O2 (0.3%). Afterwashing in PBS, sections were preincubated in blocking buffer(0.1% Triton X-100, 10% bovine serum albumin and 10% goat

serum in PBS) for 2 h at room temperature to block non-specific binding. This was followed by overnight incubation at4 °C with primary antibodies diluted in blocking buffer.Sections were incubated for 1 h at room temperature with abiotin-conjugated goat anti-rabbit IgG (1:200, Vector labora-tories, AbCys, Paris, France), diluted in blocking buffer withoutTriton X-100. They were then processed through an avidin/biotin/peroxidase complex (ABC Elite Kit, Vector Laboratories)for 1 h. Each incubation was followed by three rinses of 10 minin PBS at room temperature. Finally, sections were reacted in0.06% 3,3′ diaminobenzidine tetrahydrochloride, 0.03% NiCl2and 0.03% H2O2 in Tris–HCl buffer 0.05 M (pH 7.6). Followingdehydration in graded ethanols, the sections were clarified inxylene, cover-slipped with DPX (Fluka, Sigma-Aldrich) andexamined with a Zeiss microscope.

For fluorescent double-immunostainings, sections werepermeabilized in PBS-0.1% Triton X-100 and then preincu-bated in blocking buffer (0.1% Triton X-100, 5% bovine serumalbumin and 5% normal serum from the species of origin forthe appropriate secondary antibodies, in PBS pH 7.4) for90 min at room temperature to block non-specific binding.This was followed by overnight incubation at 4 °C withprimary antibodies diluted in blocking buffer. Sections wereincubated for 90 min at room temperature with biotin-conjugated goat anti-rabbit IgG (1:200, Vector laboratories)and TR-conjugated horse anti-mouse IgG (1:100, Vectorlaboratories) for double-stainings with mouse monoclonalantibodies. For NgR1/OMP double-labeling, sections weresequentially processed with a TRITC-conjugated mouse anti-goat Ig (1:100, Jackson Immunoresearch Lab, AbCys, Paris,France) then with the biotin-conjugated goat anti-rabbit IgG.All sections were then incubated for 90 min at roomtemperature with Alexa 488-conjugated Streptavidin (1:1000,Molecular Probes). Sections were finally mounted withVectashield mounting medium (Vector laboratories) andexamined with a Zeiss fluorescence microscope. Digitizedimages were processed for adjustment of brightness andcontrast with an image-editing software (Adobe Photoshop7.0). All double-stained sections were analyzed by confocalscanning microscopy (Leica TCS-SP2, magnification ×63,aperture 1.32–0.6, at the Centre Commun de Quantimétrie,Université Claude Bernard-Lyon1, France).

Acknowledgments

This research was supported by the Centre National de laRecherche Scientifique and Université Claude Bernard Lyon1(UMR 5020 - CNRS/UCB Lyon1). Plasmids containing themouseNgR1 cDNA or the rat NgR2 and NgR3 cDNAs were kindlyprovided by Dr. D. Hunt (Windeyer Institute, UniversityCollege of London, UK) and Dr. S. Frentzel (Novartis PharmaResearch, Basel, Switzerland) respectively. We are also grate-ful to Dr. R.J. Giger (Center for Aging and DevelopmentalBiology, University of Rochester School of Medicine andDentistry, New York, USA) for supplying NgR1, NgR2 andNgR3 antibodies. We thank Dr. Liliane Astic and Dr. DianeSaucier for the thorough and helpful reading of the manu-script and Dr. Charles A. Greer (Yale University) for preciouscomments on this work.

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