Patterning of Olfactory Sensory Connections IsMediated by Extracellular Matrix Proteins in theNerve Layer of the Olfactory Bulb

J. E. Crandall,1,2 C. Dibble,1 D. Butler,1 L. Pays,1 N. Ahmad,1 C. Kostek,1 A. W. Puschel,3

and G. A. Schwarting1,2,4

1 The Shriver Center, 200 Trapelo Road, Waltham, Massachusetts 02452

2 Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts 021144 Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115

3 Molecular Genetics Laboratory, Department of Neurochemistry, Max-Planck-Institut for BrainResearch, Deutschordenstr. 46, D-60528 Frankfurt, Germany

Received 21 April 2000; accepted 5 September 2000

ABSTRACT: In early rat embryos when axonsfrom sensory neurons first contact the olfactory bulbprimordium, lactosamine-containing glycans (LCG) aredetected on neurons that are broadly distributed withinthe olfactory epithelium, but that project axons to a veryrestricted region of the ventromedial olfactory bulb.LCG1 axons extend through channels defined by thecoexpression of galectin-1 andb2-laminin. These twoextracellular matrix molecules are differentially ex-pressed, along with semaphorin 3A, by subsets of en-sheathing cells in the ventral nerve layer of the olfactorybulb. The overlapping expression of these moleculescreates an axon-sorting domain that is capable of pro-moting and repelling subsets of olfactory axons. Specif-

ically, LCG 1 axons preferentially grow into the regionof the nerve layer that expresses high amounts of galec-tin-1, b2-laminin, and semaphorin 3A, whereas neuro-pilin-11 axons grow in a complementary pattern, avoid-ing the ventral nerve layer and projecting medially andlaterally. These studies suggest that initial patterning ofolfactory epithelium to olfactory bulb connections is, inpart, dependent on extracellular components of the em-bryonic nerve layer that mediate convergence and di-vergence of specific axon subsets.© 2000 John Wiley & Sons,

Inc. J Neurobiol 45: 195–206, 2000

Keywords:olfactory axon guidance; galectin; laminin;semaphorin; neuropilin

Physiological studies of activity in the olfactory bulb(Stewart et al., 1979; Kauer et al., 1987) as well asimmunocytochemical (Mori et al., 1985; Schwob andGottlieb, 1986; Schwarting and Crandall, 1991), his-tochemical (Key and Akeson, 1993; Treolar et al.,1996), and tract-tracing (Astic et al., 1987; Schoen-feld et al., 1994) studies have demonstrated that ax-

onal projections from broadly defined regions of theolfactory epithelium (OE) converge on spatially de-fined glomeruli in the olfactory bulb (OB). However,studies in the moth (Oland et al., 1998; Oland andTolbert, 1998) and in mice (Bulfone et al., 1998)suggest that target-related cells play an insignificantrole in olfactory axon guidance. Thus, interactionsbetween olfactory axons and glial cells or their prod-ucts along axon pathways and in the nerve layer of theOB may play an important role in olfactory axonguidance. One model for patterning connections fromthe OE to the OB early in development is to divide the

Correspondence to:Gerald A. Schwarting (GSchwarting@Shriver.org).

Contract grant sponsor: National Institutes of Health; contractgrant numbers: DC 00953 and HD 05515.© 2000 John Wiley & Sons, Inc.

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target into smaller subdivisions. This could be accom-plished by positioning positive and negative guidancecues at strategic locations within axon pathways andin the outer nerve layer of the OB. The purpose ofthese cues would be to direct subgroups of axons tospecific regions of the OB. A version of this modelhas been proposed for odorant receptor molecules.Analysis of the pattern of receptor expression byinsitu hybridization (Ressler et al., 1993; Vassar et al.,1993; Strotmann et al., 1994) suggests that subsets ofreceptor cell types are spatially restricted to four cir-cumscribed zones within the OE. Furthermore, axonprojections from these zones terminate in glomeruliwithin restricted regions of the OB (Wang et al., 1998;Lin and Ngai, 1999; O’Leary et al., 1999). However,no specific cellular or molecular mechanisms havebeen found that would restrict axons to individualzones or guide axons to a specific glomerulus withinany zone.

Odorant receptor deletion and swap experimentscombined with the use of tau-lacZ transgenic micehave suggested an instructive role for odorant recep-tors in axon guidance (Mombaerts et al., 1996). How-ever, it has been difficult to define the zones in the OBcorresponding to the four zones in the OE. Olfactorycell adhesion molecule (OCAM) and CC2 are twomarkers for large groups of axons whose trajectoriesconform to the odorant-defined compartments in theOB (Yoshihara et al., 1997; Schwarting and Crandall,1991). OCAM is expressed on neurons within re-stricted regions of the OE that project axons to re-stricted segments of the OB, excluding the dorsome-dial quadrant. CC2 is expressed on a complementaryset of neurons in the dorsomedial OE (zone 1) thatproject axons to the dorsomedial OB. However, thereare no known molecules that define boundaries be-tween zones 2, 3, and 4 in the OB.

Galectin-1 is one of very few molecules discoveredto date that may guide olfactory axons. It is a carbo-hydrate-binding protein (Cooper et al., 1991) thatspecifically interacts with lactosamine-containing gly-cans (LCGs). It is abundant in the nerve layer of theOB (Mahanthappa et al., 1994; Raabe et al., 1997)where it may interact with axons expressing LCG.Furthermore, axons that normally target the dorsome-dial OB failed to project properly in galectin-1 null,mutant mice (Puche et al., 1996). Semaphorins are afamily of axon guidance molecules that have beenextensively studied in the olfactory system, althoughno guidance function has been attributed to semaphor-ins during olfactory development. (Giger et al., 1996;Kobayashi et al., 1997; Pasterkamp et al., 1998). Onefamily member, semaphorin 3A (Sema3A; Sema-phorin Nomenclature Committee, 1999) delays the

growth of olfactory sensory fibers into the telenceph-alon in embryonic chick (Kobayashi et al., 1997).Similarly, in rats, Sema3A is thought to restrict olfac-tory axon growth but not necessarily guide axons tospecific targets (Giger et al., 1996; Pasterkamp et al.,1998). Although semaphorins are generally associatedwith repulsive activity, Sema3A can also attract den-drites (Polleux et al., 2000).

Here we describe the distribution of putative guid-ance cues in the nerve layer of the OB and the cor-responding trajectories of large groups of definedaxon subsets. LCG1 axons project to the ventrome-dial OB through an extracellular environment en-riched in galectin-1 and laminin. This projection pat-tern is established early in development and persistspostnatally, with some modifications. Furthermore,Sema3A is also localized to the extracellular matrix(ECM) of the nerve layer of the ventromedial OB andappears to divert neuropilin-11 axons to the medialand lateral OB. These contrasting activities establishthe ventral nerve layer as a critical axon sorting regionduring development of the olfactory system.

METHODS

Animals and Tissue Preparation

Timed pregnant [plug date5 embryonic day 0 (E0)] andpostnatal Sprague-Dawley rats obtained from Zivic Miller(Portersville, PA) were deeply anesthetized with a mixtureof ketamine (50 mg/kg body wt) and xylazine (10 mg/kgbody wt), and individual embryos or postnatal pups wereperfused intracardially using 4% paraformaldehyde in 0.1Mphosphate buffer, pH 7.4, or with periodate-lysine-parafor-maldehyde in 0.05M phosphate buffer (2% paraformalde-hyde, 0.075M L-lysine, and 0.214% sodium metaperiodate)in accord with the Institutional Animal Care and Use Com-mittee at the Shriver Center. The heads were postfixedovernight and then cryopreserved in phosphate-bufferedsaline (PBS), containing 30% sucrose, pH 7.4, at 4°C.Alternatively, tissue from some animals was rapidly frozenin isopentane at270°C, sectioned with a cryostat, andthaw-mounted onto coated slides (SuperFrost; Fisher Sci-entific, Pittsburgh, PA).

Antibodies

The mouse monoclonal antibody, 1B2 (IgM) (Young et al.,1981), against the LCG epitope was obtained from TheAmerican Type Culture Collection (Rockville, MD). Wehave previously identified a single glycolipid present in theolfactory system that is highly immunoreactive with the1B2 antibody (Schwarting et al., 1992); however, lightbackground immunostaining in the embryo suggests thatthere may be low levels of other glycolipids and glycopro-

196 Crandall et al.

teins that weakly cross-react with the 1B2 antibody. Thenon-neuronal cell staining within the epithelium makes itdifficult to quantify neuronal expression of LCG and todetermine whether LCG1 neurons are concentrated in spe-cific regions of the OE. We have therefore relied partially onthe positions of LCG1 axons emerging from the OE toobtain a rough idea of the position of LCG1 cells in the OE.Mouse monoclonal antibodies (IgG) against theb2-lamininchain (Hunter et al., 1989) developed by Dr. J. R. Sanes(Washington University , St. Louis), were obtained from theDevelopmental Studies Hybridoma Bank maintained by theDepartment of Pharmacology and Molecular Sciences, TheJohns Hopkins University School of Medicine, Baltimore,Maryland, and the Department of Biology, University ofIowa, Iowa City, Iowa, under contract N01-HD-2-3144from the NICHD. Goat polyclonal antiserum (IgG) againstOMP (Hartman and Margolis, 1975) was kindly providedby Dr. F. Margolis, University of Maryland. Rabbit poly-clonal antiserum (IgG) against rat galectin-1 (Cooper et al.,1991) was kindly provided by Drs. D. Cooper and S. H.Barondes, UCSF. Neuropilin-1 antibodies were obtainedfrom Oncogene Sciences (Cambridge, MA). Rabbit poly-clonal antiserum (IgG) against anti-mouse p75 (the low-affinity nerve growth factor receptor) was purchased fromChemicon (Temecula, CA).

In Situ Hybridization

DIG-labeled RNA probes for Sema3A (Pu¨schel et al., 1995)were generated according to the methods described in theBoehringer Mannheim Genius 4 kit (Indianapolis, IN).Insitu hybridization was performed on coronal cryostat sec-tions through the OE and OBs of E18 Sprague-Dawley rats.Briefly, slides were treated with proteinase K (BoehringerMannheim), treated with acetic anhydride in 0.1M trietha-nolamine for 10 min, and then dehydrated through gradedethanol solutions. Hybridization was carried out at 55–56°Cfor 15 h. Slides were washed in SSC at 55°C followed by aformamide wash at 55°C and SSC washes at 37°C. DIG-labeled RNA hybrids were reacted with alkaline phospha-tase–conjugated anti-DIG Fab antibodies (Boehringer Mann-heim). Reaction product was visualized by incubating thesections with nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP; Sigma Chemical,St. Louis, MO) overnight at room temperature for 15 h.

Combined Fluorescence In SituHybridization and Immunocytochemistry

For both cell culture and cryosection experiments, we useda protocol for fluorescence double-labeling byin situ hy-bridization and immunocytochemistry (Wanner et al.,1997). The Sema3A digoxigenin riboprobe was detectedusing a Tyramide Signal Amplification (TSA)-Direct Cy3kit (NEN, Arlington, MA). Ensheathing cells were visual-ized using the antibody to p75 and a FITC-conjugateddonkey anti-rabbit IgG (Jackson Laboratories, Bar Harbor,ME). Minor adaptations were made concerning the hybrid-

ization buffer, which contained 10 mM Tris-HCl, pH 7.5,300 mM NaCl, 1 mM EDTA, 50% deionized formamide,10% dextran sulfate, and 1% blocking reagent (BoehringerMannheim).

Immunohistochemistry andImmunofluorescence

Cryopreserved heads were rapidly frozen and sectioned atvarious thicknesses (30–60mm) with a sliding microtomein either the coronal or sagittal plane. Free-floating sectionswere incubated overnight at 4°C with primary antibodydiluted in PBS containing 1% bovine serum albumin (BSA;Sigma) and 0.2% Triton X-100. Incubation with secondaryantibody diluted in PBS containing 1% BSA was carried outfor 2 h at room temperature. For immunohistochemistry,biotinylated secondary antibodies (Jackson ImmunoRe-search Laboratories, West Grove, PA) were used in combi-nation with avidin-biotin-horseradish peroxidase (Vec-tastain standard ABC kit; Vector Laboratories, Burlington,CA) and visualized with a diaminobenzidine-nickel reac-tion. For immunofluorescence, FITC- or Cy3-conjugatedsecondary antibodies (Jackson ImmunoResearch Laborato-ries) were used, or in some cases biotinylated secondaryantibodies were used in combination with Cy3-conjugatedStreptavidin.

Cell Culture

Patterned substrates were produced on glass coverslips us-ing a silicone matrix as previously described (Vielmetter etal., 1990; Wang and Anderson, 1997). The first stripe wasproduced by injecting laminin (75 mg/mL) into the openchannels of the matrix placed on a coverslip. After 1 h at37°C, the channels were washed with PBS and a 10 mg/mLsolution of purified, recombinant, Flag-tagged, Sema3A(Klosterman et al., 1998) harvested from HEK 293 cellsupernatants was injected into the channels. This concen-tration was used after dose–response experiments showedthat no axon turning activity could be demonstrated in thisassay when less than 2–3mg/mL of purified Sema3A wasused. After 1 h at37°C, the channels were washed with PBSand a 2 mg/mL solution of Texas Red-BSA (Sigma) wasinjected to block nonspecific binding and to identify thestripe by fluorescence microscopy. After washing with PBS,the matrix was removed and the coverslip was incubatedwith laminin (25 mg/mL) for 1 h at 37°C. For controlcoverslips, the addition of Sema3A was eliminated. DRGsand olfactory sensory neurons from E15 rats were dissoci-ated in serum-free neurobasal medium (Gibco BRL Labo-ratories, Grand Island, NY) and plated onto freshly preparedcoverslips. After 48 h, the cultures were fixed in 4% para-formaldehyde and stained with antibodies to NCAM fol-lowed by fluorescein-conjugated secondary antibodies. Forolfactory cultures, cell density was empirically determinedso that between 2 and 10 cell aggregates were plated oneach coverslip. Coverslips were prepared fresh on the day ofcell culture. Two control and two experimental coverslips

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were used in each experiment. Maintaining a ratio of lami-nin in the first and second stripes at;3:1 was important andwas reevaluated when a new batch of laminin was pur-chased. Initially, slides were evaluated blind for attraction/repulsion. Only when both control coverslips were correctlyidentified as having no axon turning activity, were experi-mental coverslips evaluated. In all cases in which a prepa-ration of purified Sema3A was shown to repel DRG axonsin this assay, olfactory axons were also repelled.

RESULTS

Prenatal Expression of LCG

Although small numbers of neurons in the OE andtheir axons expressed LCG at E14 in the rat, not untilE15 did a projection pattern emerge within the OBthat remained unchanged until birth. Figure 1 showsthe convergence of LCG1 axons to the ventromedialnerve layer of the OB in the coronal plane [Fig. 1(A)].At E18, LCG1 projections were also restricted in therostrocaudal plane where they were concentrated inthe nerve layer of the central OB, but did not extendto the rostral or caudal limits of the OB [Fig. 1(B)].

In contrast to the strictly demarcated pattern in theOB, virtually all regions of the developing OE con-tained LCG1 neurons. Figure 1(C–H) represents thelocation and relative density of LCG1 neurons at E18in six sections [1(C) is most rostral, 1(H) is mostcaudal] of the OE. Only the most lateral regions of theOE contained few or no LCG1 neurons. This patternconsisted of neurons scattered throughout the OE, butwith axons that projected predominantly to the ven-tromedial OB, suggested that the outgrowth of LCG1

axons was restricted along pathways to the ventralnerve layer of the OB. Indeed, there appeared to beregions of the nerve layer from which LCG1 fiberswere excluded or repelled [arrowheads in Fig.1(A,B)], adjacent to other regions of the nerve layerthat were densely populated with LCG1 fibers.

Postnatal Expression of LCG

Analysis of LCG expression in late embryonic andearly postnatal development revealed that the overallpattern in the OB remained quite similar to the patternseen from E15 to E18. The major change was theaddition of LCG1 axons terminating in the rostral anddorsal OB. To determine whether there was a patternto LCG1 axon targeting, we mapped the positions ofLCG1 glomeruli in postnatal day 6 (P6) animals.Results suggested that the positions of LCG1 glomer-uli were spatially regulated (Fig. 2). First, preciselymatched coronal sections through both OBs at P6

showed that LCG1 glomeruli formed mirror imagepatterns at all rostrocaudal levels. Second, mappingthe OB for LCG immunoreactivity in P6 littermatesdemonstrated that glomeruli reactive with LCG anti-bodies in one animal were likely to be LCG1 in allanimals. Thus, new LCG1 neurons at different agesthroughout different zones in the OE probably usedconserved guidance cues to find targets in the OB.The number, distribution, time of initial onset, andpattern of projections of LCG1 neurons were invari-able from animal to animal at this age. Figure 3 showsan example of the broad distribution of LCG1 neu-rons in the OE at P6. LCG1 neurons were found athighest density along the septum and in endoturbi-nates II and III, at moderate density in the dorsome-dial OE and at lowest density in the ventrolateral OE.LCG1 neurons were almost always located in the

Figure 1 LCG1 axons converge onto a small ventrome-dial target in the embryonic OB. At E18 (A), in a coronalsection through the olfactory epithelium (oe) and olfactorybulb (ob), LCG is expressed on axons that extend into thenerve layer of the ventral and medial OB (arrows), but isabsent from the lateral and dorsal OB. LCG1 axons areconspicuously absent from a region of the ventral nervelayer (arrowhead). At E18 (B), in a parasagittal sectionthrough the OE and OB, LCG is expressed on axons thatextend into the nerve layer of the ventral OB (arrows), butis absent from axons in the caudal, rostral, and dorsal OB.LCG1 axons appear to avoid the caudal nerve layer (arrow-head). Scale bar5 200mm. The positions of LCG1 sensoryneurons are represented in reconstructed coronal sections ofthe E18 OE at different rostrocaudal planes of the nasalcavity (C–G). Regions of the OE with the highest density ofLCG1 neurons (5–10 neurons/100mm) are dark gray. Re-gions of the OE with a moderate density of LCG1 neurons(1–4 neurons/100mm) are light gray. LCG negative regionsof the OE (,1 neuron/100mm) are white. In (D), (G), and(H), 2 and 3 denote endoturbinates; II, III, and IV denoteectoturbinates.

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outermost layer of neurons in the OE, suggesting thatthey represent a subset of the most mature OE neu-rons.

Extracellular Matrix of the EmbryonicOlfactory System

At E18, b2-laminin and galectin-1 colocalized in theECM between the OE and OB (Fig. 4). Galectin-1was expressed in the lamina propria adjacent to theOE and was also expressed along channel-like struc-tures leading from the OE to the OB [Fig. 4(A)]. Theoverall pattern of galectin-1 in the nerve layer of theOB was characterized by alternating patches of highand low expression. At higher magnification, confocalimages ofb2-laminin [Fig. 4(B)] and galectin-1 [Fig.4(C)] in the nerve pathways revealed thatb2-lamininwas prominent at galectin-1 borders and at galectin

surfaces. This was most obvious whereb2-laminin isexpressed on the surface of galectin-1–defined chan-nels [Fig. 4(D)].

In addition to the coexpression of galectin-1 andb2-laminin outlining olfactory axon pathways, a sim-ilar spatial compartmentalization was shared by thesetwo ECM proteins within the developing nerve layer.In coronal sections, galectin-1 [arrow in Fig. 4(A)]was concentrated in the outer nerve layer of the ven-

Figure 3 LCG1 neurons are distributed throughout multipleOE zones. At P6, in a coronal section (A), LCG1 neurons arevisible in most areas of the olfactory epithelium (oe). Most ofthe immunoreactive neurons (epithelial regions between ar-rows) are located in a band that extends across several turbi-nates from the septum (s) to ectoturbinates II and III. Thedorsomedial (open arrow) region of the OE contains manyfewer LCG1 neurons, whereas the ventrolateral (arrowheads)region contains almost none. LCG1 neurons are almost alwayspositioned near the luminal surface of the OE (B), suggestingthat they are among the most mature neurons in the OE (nc,nasal cavity). Medial is to the right; dorsal is up. Scale bars5 500mm (A) and 100mm (B).

Figure 2 LCG is expressed invariably in a unique subsetof glomeruli. At P6, the pattern of LCG expression wasexamined in coronal sections. In sections matched for right/left symmetry (A), LCG1 glomeruli are present in the samelocations in the ventromedial segment of each OB (largearrows). Scattered glomeruli in the dorsal half of the OB(small arrows) also reveal a symmetrical pattern. Matchingcoronal sections from the OBs of littermates (B,C) revealsthat the positions of LCG1 glomeruli are nearly identicalventromedially (arrows). It appears that the positions ofLCG1 neurons and glomeruli are tightly regulated in timeand space. Scale bar5 500 mm.

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tromedial OB. This region of high galectin-1 expres-sion was situated between two large regions of thenerve layer that were nearly devoid of galectin-1; oneis in the ventrolateral nerve layer, the other is in themedial nerve layer [arrowheads in Fig. 4(A)].

In axon pathways, LCG1 axon bundles extendedthrough channels defined by galectin-1 expression(Fig. 5) and into the region of the nerve layer of theventromedial OB that heavily expressed galectin-1.However, there were some galectin/laminin channels[Fig. 5(A)] that were not utilized by LCG1 axons. Inthose channels that were traversed by LCG1 pro-cesses, confocal double-labeled images reveal thatLCG1 axons rarely crossed borders defined by galec-tin-1 expression [Fig. 5(B), 5(C), and 5(D)]. Thus,although the ventromedial nerve layer expressed mol-ecules that permit the convergence of LCG1 fibers,not all galectin/laminin channels were restricted foruse by LCG1 axons.

Permissive and Nonpermissive Cues inthe Ventromedial Nerve Layer

The ventromedial nerve layer of the olfactory bulb isalso characterized by the expression of other mole-cules that may be involved in the guidance of olfac-tory axons. One of these is p75, the low-affinity nervegrowth factor receptor that is often used as a markerfor ensheathing cells (Barnett et al., 1993; Gong et al.,1994; see reviews, Doucette, 1984; Ramon-Cueto andValverde, 1995). p75 was expressed along axon path-ways and within the ventral nerve layer, in virtuallythe same pattern as galectin-1 andb2-laminin. Theventromedial nerve layer expressed p75 heavily,whereas two adjacent regions of the nerve layer ex-pressed low amounts of this cell surface molecule[Fig. 6(D)]. It was recently shown that p75 is largelyconfined to the outer nerve layer, whereas cells in theinner nerve layer express NPY (Ubink and Ho¨kfelt,

Figure 4 Galectin-1 andb2-laminin are coexpressed in the extracellular matrix of axon pathways.In a 40-mm-thick coronal section at E18, galectin-1 (A) is expressed in the lamina propria anddefines channel-like structures leading from the OE to the OB. There is a high concentration ofgalectin-1 in the ventromedial nerve layer of the OB (arrow), bordered by two areas of very lowconcentration of galectin-1 (arrowheads). At high magnification (of boxed area in A), 1-mm-thickconfocal images show thatb2-laminin (B) and galectin-1 (C) form boundaries outlining axonpathways. Double-label immunofluorescence (D) reveals a pattern in whichb2-laminin is oftenexpressed on the surface of galectin-1–defined boundaries (arrows). Scale bars5 200mm (A) and50 mm (B–D).

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2000). Taken together, these results clearly indicatethe molecular heterogeneity in the developing olfac-tory nerve layer. In contrast, Nissl staining, showingthe distribution of cell bodies, suggests that glial cellsare uniformly distributed throughout the entire nervelayer (Kafitz and Greer, 1999).

In addition, the ventromedial nerve layer expresseda known axon guidance protein, Sema3A (first de-scribed in chick as collapsin-1; Luo et al., 1993).Sema3A expression was more restricted than the otherECM proteins and within the nerve layer was confinedto the ventromedial OB [Fig. 6(C)]. This can be seenon serial sections individually reacted with antibodiesto galectin-1 and p75 and with riboprobes for Sema3A(Fig. 6). At higher magnification, a region in the outernerve layer that expressed high levels of Sema3A[Fig. 6(E)] overlapped extensively with a region ofthe outer nerve layer that was p751 [Fig. 6(F)]. Wehave also carried out double-labelin situ hybridiza-tion for Sema3A and immunocytochemistry for p75

on tissue sections and primary ensheathing cellsgrown in vitro, which demonstrated that Sema3A wasexpressed on a subset of p751 ensheathing cells (datanot shown). Sema3A can repel and attract neuritesthat express neuropilin-1 as the ligand-binding sub-unit of the receptor (He and Tessier-Lavigne, 1997;Kolodkin et al., 1997; Takahashi et al., 1999; Polleuxet al., 2000). As shown in Figure 6(B), neuropilin-11

axons extended medially and laterally around the re-gion of the ventromedial nerve layer that expressedSema3A.

The trajectories of neuropilin-11 olfactory axonsin the embryo (Fig. 7) were similar to the pattern ofneuropilin-11 glomeruli in adults (Pasterkamp et al.,1998). At the rostral tip of the olfactory bulb, neuro-pilin-11 axons extended along the lateral surface ofthe OB [Fig. 7(A)]. In the central OB, neuropilin-11

axons grew medially and laterally [Fig. 7(B)], but inthe caudal OB, neuropilin-11 axons grew only medi-ally [Fig. 7(C)]. Thus, not only were these axons

Figure 5 LCG1 axons are restricted by galectin-1 channels. In a double-labeled 1-mm-thickconfocal image of a coronal section through the OE and OB at E18, LCG1 fascicles (red in A)extend through channels defined in part by galectin-1 (green in A), predominantly to the nerve layer(nl) of the ventral OB (arrow). At high magnifications of the boxed area in (A), galectin-1 (B) isdeposited at the borders of axon pathways and LCG1 axons (C) form large bundles as they traversethese pathways. Double-label immunofluorescence (D) demonstrates that LCG1 axon bundlesrarely cross boundaries defined by the expression of galectin-1. Scale bars5 200 mm (A) and 50mm (B–D).

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apparently repelled from the ventral nerve layer, theywere also restricted to the rostrolateral and caudome-dial segments of the developing OB. When comparedin serial sections, LCG1 axons [Fig. 7(D)] and neu-ropilin-11 axons [Fig. 7(B)] grew into complemen-tary regions of the nerve layer.

The complementary pattern of expression ofSema3A and neuropilin-1 suggests that Sema3A mayact as a repellent for neuropilin-11 olfactory neurons.To test this, we grew olfactory sensory neurons onpatterned substrates containing alternating stripes oflaminin and purified Flag-tagged Sema3A (Fig. 8). Asnegative and positive controls, dissociated DRG neu-rons from E15 rats were grown on striped substrates.When no Sema3A was present in either stripe, DRGaxons randomly grew on the coverslips [Fig. 8(A)],demonstrating that there were no physical or molec-

ular barriers at stripe borders. When one stripe, (1),contained Sema3A, DRG cell bodies and axons re-mained on the Sema3A-negative stripe [Fig. 8(B)]. Adose–response curve showed that no activity was ap-parent when the concentration of Sema3A was,2mg/mL. The results shown in Figure 8 are represen-tative of five experiments performed using three sep-arate batches of purified Sema3A at 10mg/mL.

Viability issues limited our ability to grow fullydissociated olfactory sensory neuron cultures. There-fore, we used semidissociated olfactory epithelial cul-tures. They consisted of mixed clusters of nonneuro-nal cells and neurons, which extended axons for longdistances away from the cell cluster. Once axons hadgrown;50 mm away from the cell cluster, they onlymade contact with the substrate-coated coverslip. Un-like DRG cultures, olfactory axons fasciculated ex-

Figure 7 Neuropilin-11 and LCG1 axon pathways are com-plementary. In coronal sections of the E18 olfactory bulb,neuropilin-11 axons extend laterally (arrow in A) in the rostralOB and medially (arrow in C) in the caudal OB. In serialsections through the central OB, neuropilin-11 axons growmedially and laterally (arrows in B) but avoid the ventral nervelayer (open arrowhead in B), whereas LCG1 axons remain inthe ventral nerve layer of the OB (arrow in D). Medial is to theright; dorsal is up. Scale bar5 200mm.

Figure 6 Ensheathing cells in the ventromedial nerve layerexpress axon guidance molecules. Immunocytochemistry(A,B,D,F) and in situ hybridization (C,E) on 20-mm-thickserial sections from the E18 olfactory bulb show that galectin-1(A), p75 (D,F), and Sema3A (C,E) expression by ensheathingcells overlaps predominantly in the nerve layer of the ventro-medial OB (arrows). Neuropilin-11 axons (open arrows in B)avoid the ventromedial nerve layer and instead grow mediallyand laterally. A higher magnification of the nerve layer showsthat Sema3A (E) and p75 (F) are expressed primarily in theouter nerve layer. Medial is to the right; dorsal is up. Dashesindicate the outer border of the nerve layer. An asterisk islocated between the same two capillaries in (E) and (F). Scalebar5 200mm (A–D) and 67mm (E,F).

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tensively, making it nearly impossible to determinethe number or percentage of axons that cross bound-aries. Also unlike DRG axons, a significant number ofolfactory axons grew onto and off of Sema3A-con-taining stripes. In contrast, a large number of olfactoryaxons preferentially grew along Sema3A-negativestripes and did not cross Sema3A-positive borders[Fig. 8(D)]. A higher magnification image of olfactoryaxons [Fig. 8(E,F)] shows that axon bundles stayed afew micrometers away from the Sema3A-containingsubstrate. This pattern of growth was not seen oncontrol stripes [Fig. 8(C)]. Because of extensive fas-ciculation, it was not possible to determine the per-centage of olfactory axons that were repelled bySema3A. However, in general, Sema3A appeared torepel subsets of olfactory sensory neuronsin vitro.

DISCUSSION

LCG1 Axons Converge into theVentromedial Olfactory Bulb

Studies reported here demonstrate that LCG1 neuronsextended their axons consistently from a broad region ofthe OE to a restricted target region of the OB. Thisstereotypical axon convergence was seen very early indevelopment as LCG1 axons terminate in the ventrome-dial nerve layer. The pattern continued in adult animalsas LCG1 glomeruli were characteristically detected inthe same positions of the OB. The positions of LCG1

glomeruli formed mirror images in coronal sectionsfrom the same animal and generated nearly identicalpatterns in the OB from animal to animal. This type of

Figure 8 Sema3A repels a subset of olfactory axonsin vitro. Dorsal root ganglion (DRG) neuronsand olfactory sensory neurons from E15 rats were grown on striped substrates and visualized byimmunofluorescence staining with antibodies to NCAM. On control stripes that contained equalamounts of laminin, DRG axons (arrowheads in A) randomly crossed control stripe borders. Onalternating striped substrates containing Sema3A (1) and DRG cell bodies and processes (B) growexclusively on the stripes that contain no Sema3A. Semidissociated olfactory sensory neuronsextend processes randomly across control stripe borders (arrowheads in C). On alternating Sema3Astriped substrates many olfactory sensory axons grow onto and across Sema3A containing stripes(arrowhead in D). After they have grown away from the explant, axon bundles (arrows in D) extendlong processes on the Sema3A-negative substrate and turn away from Sema3A-positive borders. Athigher magnification (E,F), large fascicles turn gradually as they approach Sema3A-containingsubstrates. These bundles do not cross onto the Sema3A-containing substrate, but remain fascicu-lated for long distances. Scale bar5 50 mm (A–D) and 15mm (E,F).

Olfactory Axon Guidance 203

pattern can only be sustained if there are mechanismsavailable that can guide defined axons to their targets,based on molecular interactions. In this instance, such amechanism is possible because LCG on olfactory neu-ron cell surfaces interacts specifically with galectin-1invitro (Mahanthappa et al., 1994). Galectin-1, an extra-cellular matrix molecule expressed in the lamina propriaand nerve layer, is one of a few molecules shown toparticipate in olfactory axon guidance (Puche et al.,1996; Tenne-Brown et al., 1998).

Npn-1 Axons Diverge at theVentromedial Olfactory Bulb

We also showed here that widely dispersed neuropi-lin-11 neurons projected axons that diverged as theyencountered the nerve layer of the ventromedial OB.The fact that Sema3A is heavily expressed in theventromedial nerve layer and can repel olfactory ax-ons that express neuropilin-1 provides a simple mech-anistic explanation for this phenomenon. The signif-icance of these findings is that it establishes theventromedial nerve layer of the OB as a unique mo-lecular environment. Galectin-1, p75, and Sema3Aare all expressed by ensheathing cells in the ventro-medial nerve layer. Two adjacent regions, one medial,the other ventral, express low levels of these threemolecules. The fact that LCG1 and neuropilin-11

axons grow into complementary regions of the nervelayer, converging and diverging precisely in this ven-tromedial domain, suggests that the molecular envi-ronment at that location contains both positive andnegative axon guidance cues.

In some areas of the embryonic nerve layer, therewere relatively sharp boundaries for LCG1 axontracts (see Fig. 1). Most often, this did not appear tobe associated with expression of any known ECMproteins. Rather, it appeared that LCG1 axons did notgrow into areas that were heavily populated withneuropilin-11 axons, suggesting that axon competi-tion for delimiting permissive substrates may play arole in restricting LCG1 axons to ventromedial tar-gets. A similar repulsion–competition model has beenproposed for ephrin function in retinocollicular map-ping (Feldheim et al., 2000).

Galectin-1 and b2-laminin areCoexpressed in the Nerve Layer

It is still not clear if galectin-1 guides LCG1 axons orif it merely supports axon extension. Here, we showthat galectin-1 specifically associated with laminins inthe nerve layer of the OB. LCG1 axon bundles lead-ing from the OE to the ventromedial OB were sur-

rounded by laminin/galectin channels, suggesting thatboundaries of laminin/galectin defined pathwaysthrough which LCG1 axons prefer to grow. Confocaldouble-label immunofluorescence studies suggest thatlaminin forms a layer on the surface of galectin-1 inthe ECM. It appears that gaps in the expression ofgalectin-1 define channels through which LCG1 ax-ons grow and that the inner surface of these channelsis coated with laminin. Thus, LCG1 axons may nevercome in direct contact with galectin-1, which maysimply act as a support for laminin or other permissiveor nonpermissive substrates.

Another question relates to the expansion of LCG1

targets to the rostral OB. Beginning around birth, anadditional group of LCG1 glomeruli are detected inthe rostral and dorsal OB. The new group of LCG1

axons would be expected to recognize similar guid-ance cues as the initial group of LCG1 axons, but ifthe spatial expression of those cues is altered overtime, the target could shift. Interestingly, there is aspatio-temporal pattern of ensheathing cell differenti-ation in the developing rat olfactory system (Astic etal., 1998); thus, maturational differences betweenglial cells in different locations of the nerve layercould produce environments that contribute to fasciclereorganization. Spatio-temporal changes in the ex-pression patterns of ECM molecules such as laminin,fibronectin, chondroitin sulfate proteoglycan, tenas-cin, and type IV collagen (Gonzalez et al., 1993;Gong and Shipley, 1996; Julliard and Hartmann,1998) could also contribute to changes in olfactoryaxon pathways (Kafitz and Greer, 1998).

Ensheathing Cells Express GuidanceCues

The identification of glial cells presenting both per-missive and nonpermissive guidance cues may beunique. A number of glial-derived axon repellentshave been described, such as myelin-associated neu-rite growth inhibitors (Caroni and Schwab, 1988). Themajority of these molecules, including chondroitinsulfate proteoglycans and tenascin, are usually non-permissive to all axons, not just a subset (e.g., Gonza-lez et al., 1993). Many of the other recently describedchemorepellents, such as semaphorins, netrins, andEph receptors (O’Leary et al., 1999), are usually tar-get-derived, and most often are the product of neu-rons. One exception is Sema3A, which is expressed inthe olfactory system, not only in mitral and tuftedcells, but also in non-neuronal cells along axon path-ways (Giger et al., 1996; Pasterkamp et al., 1998).The homologous molecule collapsin-1 is expressed inthe nerve layer of the developing chick OB and delays

204 Crandall et al.

the growth of olfactory axons into the OB primordium(Kobayashi et al., 1997). Here we show that Sema3Ais expressed in a subset of ensheathing cells in theventromedial nerve layer of the OB in a manner thatcould restrict the growth of neuropilin-11 axons.These findings in the developing olfactory system areconsistent with and provide additional support for animportant role of semaphorin–neuropilin interactionsin establishing early axon projections in the develop-ing nervous system (Polleux et al., 1998).

We thank Peter Elenbaas for excellent technical assis-tance.

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