Schwob 2002 the Anatomical Record

FEATURE ARTICLE

Neural Regeneration and the Peripheral OlfactorySystemJAMES E. SCHWOB*

The peripheral olfactory system is able to recover after injury, i.e., the olfactory epithelium reconstitutes, the olfactorynerve regenerates, and the olfactory bulb is reinnervated, with a facility that is unique within the mammalian nervoussystem. Cell renewal in the epithelium is directed to replace neurons when they die in normal animals and does so atan accelerated pace after damage to the olfactory nerve. Neurogenesis persists because neuron-competentprogenitor cells, including transit amplifying and immediate neuronal precursors, are maintained within thepopulation of globose basal cells. Notwithstanding events in the neuron-depleted epithelium, the death of bothnon-neuronal cells and neurons directs multipotent globose basal cell progenitors, to give rise individually tosustentacular cells and horizontal basal cells as well as neurons. Multiple growth factors, including TGF-!, FGF2,BMPs, and TGF-"s, are likely to be central in regulating choice points in epitheliopoiesis. Reinnervation of the bulbis rapid and robust. When the nerve is left undisturbed, i.e., by lesioning the epithelium directly, the projection of thereconstituted epithelium onto the bulb is restored to near-normal with respect to rhinotopy and in the targeting ofodorant receptor-defined neuronal classes to small clusters of glomeruli in the bulb. However, at its ultimate level, i.e.,the convergence of axons expressing the same odorant receptor onto one or a few glomeruli, specificity is notrestored unless a substantial number of fibers of the same type are spared. Rather, odorant receptor-definedsubclasses of neurons innervate an excessive number of glomeruli in the rough vicinity of their original glomerulartargets. Anat Rec (New Anat) 269:33–49, 2002. © 2002 Wiley-Liss, Inc.

KEY WORDS: axon specificity; development; epitheliopoiesis; growth factors; neuroscience; nerve transection; stem cell,neural; neurogenesis; odorant receptors

INTRODUCTIONThe olfactory system is unusualamong sensory systems in several re-

spects. Most notably, the receptor el-ements that subserve the olfactorysense are embedded in the olfactoryepithelium, which lines a part of thenasal cavity, and are in direct contactwith the airborne environment, asthey must be to transduce volatilechemical stimuli (Farbman, 1992).Furthermore, the primary sensorycells are bona fide neurons with pro-jections into the central nervous sys-tem (CNS) that offer a potential routeof transport of infectious agents orother materials from the external en-vironment (Bodian and Howe, 1941;DeLorenzo, 1970; Monath et al.,1983). As a consequence of their rela-tively unprotected position in the na-sal cavity, the cells of the olfactoryepithelium can be damaged easily byexposure to toxins, infectious agents,or trauma. Most significantly, the pri-mary olfactory projection, i.e., the ol-factory epithelium and its projectionby means of the olfactory nerve ontoits synaptic target in the CNS, the ol-factory bulb, is an exception to the

general rule that the nervous systemrepairs itself only very poorly after in-jury. Indeed, the remarkable capacityof the olfactory system for recoveryafter injury helps maintain criticalsensory function, despite the system’svulnerability to damage.

Investigative evaluation of regener-ation in the primary olfactory projec-tion dates from the middle of the lastcentury and includes the work of Na-gahara, which demonstrated regener-ation after experimental nerve injury(Nagahara, 1940). Subsequent studiesover the next two decades demon-strated the destruction and reconsti-tution of the epithelium after irriga-tion with zinc sulfate (ZnSO4), whichis directly toxic to the epithelium(Schultz, 1941; Smith, 1951; Schultz,1960; Mulvaney and Heist, 1971;Matulionis, 1975, 1976; Harding et al.,1978; Burd, 1993; Thompson et al.,2000). Research in this area was invig-orated by observations that the epi-thelium contains neurons of increas-ing maturity as one proceeds from

Dr. Schwob is a developmental neurobi-ologist whose interest in the olfactorysystem as a model for understandingfundamental events in neural develop-ment was piqued by the demonstrationsby Graziadei and Moulton in the 1970sthat neurogenesis persists in the olfac-tory periphery throughout life. He com-pleted M.D. and Ph.D. at WashingtonUniversity in St. Louis, studying the de-velopment of axonal connectivity in thecentral olfactory system with Dr. Jo-seph L. Price. During postdoctoral workwith Dr. David I. Gottlieb, also at Wash-ington University, he analyzed olfactorysensory neuron molecular phenotype.Formerly Chair of the Department ofCell and Developmental Biology atSUNY Upstate Medical in Syracuse, herecently became Professor and Chair ofAnatomy and Cellular Biology at TuftsUniversity School of Medicine.Grant sponsor: NIH; Grant number: R01DC00467; Grant number: R01 DC02167.*Correspondence to: James E. Schwob,Department of Anatomy and Cellular Bi-ology, Tufts University School of Medi-cine, 136 Harrison Avenue, Boston, MA02111.E-mail: [email protected]

THE ANATOMICAL RECORD (NEW ANAT.) 269:33–49, 2002DOI 10.1002/AR.10047

© 2002 Wiley-Liss, Inc.

basal to apical in the normal olfactoryepithelium, and by data obtained byusing the [3H]thymidine technique forassessing cellular proliferation (An-dres, 1965; Moulton et al., 1970; Gra-ziadei and Metcalf, 1971; Graziadei,1973; Moulton, 1974). The latter ap-proach established directly that the ol-factory epithelium retains a popula-tion of proliferating progenitor cellsin the basal layers of the epitheliumthroughout life, whose daughters canbe “chased” apicalward with the pas-sage of time into the neuronal com-partment of the epithelium (Graziadeiand Graziadei, 1979).

The persistence of progenitor ele-ments throughout adulthood providesa means of accomplishing replace-ment of olfactory neurons after theirexperimentally induced destructionby knife cut or lavage with a coagulanttoxin such as ZnSO4. Thus, the notionwas engendered that the olfactoryneuroepithelium undergoes a consti-tutive, piecemeal turnover of the neu-ronal population analogous to cellularreplacement in other non-neural epi-thelia, as well as a wholesale reconsti-tution of that population after lesionas a kind of wound healing (Moulton,1975; Graziadei and Monti Graziadei,1978). The scope of the problem fur-ther expanded with the discovery ofthe large family of odorant receptor(OR) genes, the data suggesting selec-tive expression of only one allele ofonly one OR gene by individual neu-rons in a spatially restricted manner,and the documentation of the exquis-ite specificity of axon targetingwhereby axons from neurons express-ing the same OR converge onto onepair of target glomeruli of the 2000 orso glomeruli in the bulb (Buck andAxel, 1991; Ngai et al., 1993; Ressler etal., 1993, 1994; Vassar et al., 1993,1994; Chess et al., 1994; Strotmann etal., 1994a,b, 1995a,b, 1996, 2000;Mombaerts et al., 1996). Understand-ing the extent to which specificity ofOR expression and axon targeting re-emerge during regeneration is crucialfor defining the capacity for recover-ing sensory function after injury andmaintaining perceptual stability dur-ing constitutive turnover.

As it has become evident that neuralprogenitor elements persist and areactive in the adult CNS (Gage et al.,1995), albeit to a limited degree, the

retention of a robust capacity for re-pair and replacement in the olfactorysystem has become less of a biologicalcuriosity and more of an exemplar ofa fundamental capacity for adult neu-rogenesis. Thus, one may hope that amore detailed understanding of theevents of epithelial reconstitution willinform our understanding of cell gen-eration in the CNS during develop-ment and in maturity and lead tostrategies for making replacement ofCNS neurons more robust. Accord-ingly, my purpose is to review what isknown regarding the process of epi-thelial reconstitution in the olfactorysystem and, to a lesser extent, the re-innervation of the olfactory bulb. Myintention is to highlight the cellularand molecular regulation of neuronalregeneration and to outline strategiesfor future investigation. First, how-ever, it is necessary to describe the

constituent cell types and the phe-nomenology of turnover and regener-ation in the olfactory system.

CELLULAR CONSTITUENTS OFTHE PERIPHERAL OLFACTORYSYSTEMThe olfactory mucosa, consisting ofthe olfactory neuroepithelium and theunderlying lamina propria, lines theposterodorsal nasal cavity in terres-trial mammals (Farbman, 1992). Theepithelium is composed of a limitednumber of cell types whose somataare arranged in a roughly laminar pat-tern (Fig. 1). From the apical surfaceto the basal lamina, they are susten-tacular (Sus) cells, mature olfactorysensory neurons (OSNs), immatureOSNs, globose basal cells (GBCs), andhorizontal basal cells (HBCs). In addi-tion, Bowman’s glands/ducts (BG/D)

extend from the lamina propriathrough the epithelium to dischargecontents at the apical surface (see Ta-ble 1 for a complete list of abbrevia-tions). Finally, the fascicles of the ol-factory nerve, in which the axons ofthe OSNs project to the olfactory bulbaccompanied by ensheathing glia, runroughly anterior to posterior in thelamina propria converging progres-sively to a small number of large bun-dles that pass through the foramina ofthe cribriform plate.

The Sus cell is a non-neuronal sup-porting cell capped by microvilli,rather than the cilia that are typical ofcolumnar respiratory epithelial cells,and contains abundant endoplasmicreticulum (Farbman, 1992). Sus cellsexpress cytokeratins 8 and 18, whichare generally found in simple epithe-lial cells, including those in respira-tory epithelium (Schwob et al., 1995).Sus cells express multiple cytochromeP450s and other biotransformationenzymes at levels higher than in liver,suggesting that Sus cells serve a detox-ification function (Ding and Coon,1988; Chen et al., 1992). Sus cells alsophagocytose dead neurons when thelatter die at a rapid clip (Suzuki et al.,1995, 1996). The low rate of Sus cellproliferation has been interpreted asindicating slow turnover and self-re-placement, and/or an accommodationto the slow growth of the olfactoryepithelium in rodents (Graziadei andGraziadei, 1979; Weiler and Farbman,1998). Microvillar cells, a second typeof supporting cell, lack the abundantendoplasmic reticulum of Sus cellsand express distinct antigens (Carr etal., 1991).

Mature OSNs are bipolar in shape:an apical dendrite ends in a knob thatelaborates 12 or more cilia splayingout over the surface of the epithelium,and a thin, unmyelinated axon exitsthe epithelium basally to join with fas-cicles of the olfactory nerve runningto the bulb (Farbman, 1992). Physio-logical data and analyses of the den-sity of intramembranous particlessuggest that sensory transduction oc-curs in the cilia (Farbman, 1992). Themature neurons are marked by the ex-pression of the olfactory marker pro-tein (OMP), and components of thesensory signal transduction cascade(Monti Graziadei et al., 1977). Deep tothe band of mature neurons sit imma-

The remarkablecapacity of the olfactorysystem for recovery after

injury helps maintaincritical sensory function

despite the system’svulnerability to damage.

34 THE ANATOMICAL RECORD (NEW ANAT.) FEATURE ARTICLE

ture OSNs, which express GAP-43 at ahigh level but not OMP, and have notyet extended cilia (Verhaagen et al.,1989; Meiri et al., 1991; Schwob et al.,

1992). The ratio of mature to imma-ture neurons varies through the life ofthe animal. The epithelium is com-posed of mainly immature olfactory

neurons early in the development ofthe olfactory system, during the pe-riod immediately after a reversible le-sion of the olfactory epithelium, andover the long-term as a consequenceof the absence of the synaptic target(Verhaagen et al., 1989, 1990; Schwobet al., 1992; Schwob et al., 1995; Looet al., 1996). Based on these and otherdata, the time required for a newlyborn neuron to make the transition tomaturity is approximately 1 week af-ter the last mitosis (Miragall andMonti Graziadei, 1982; Schwob et al.,1992).

The basal cells of the epithelium aredivided into two categories (Andres,1965; Graziadei and Graziadei, 1979).GBCs are simple, round cells withscant cytoplasm. Several functionallyanonymous markers have been iden-tified that label GBCs, but are not lim-ited to them (Goldstein and Schwob,1996; Goldstein et al., 1997). GBCs ex-hibit a high proliferative rate, suchthat the vast majority of cells labeled

Figure 1. Cellular constituents and progenitor-progeny relationships in the normal olfactory epithelium and after neuronal depletion. Eachclass of non-neuronal cell resides within its own separate lineage in this context. However, a category of globose basal cell (GBC) that ismultipotent is mitotically active even when only neurons are made. Other than general GBC markers, for example, immunoreactivity withthe monoclonal antibody GBC-2, no marker is known to identify the class of multipotent progenitors specifically. In contrast, the transitamplifying and immediate neuronal precursor classes express MASH1 and neurogenin-1 (Ngn-1), respectively. BD, Bowman’s duct; BG,Bowman’s gland; GBCmpp, multipotent progenitor subclass of GBC; GBCta, transit amplifying subclass of GBC; GBCinp, immediate neuronalprecursor subclass of GBC; Sus, sustentacular cell; HBC, horizontal basal cell; OSNi, immature olfactory sensory neuron (growth-associatedprotein-43–expressing, nonciliated, axons tipped by growth cones); OSNm, mature OSN (olfactory marker protein [OMP]-expressing,ciliated, synaptically connected to bulb).

TABLE 1. Abbreviations for cell types, factors, receptors, and reagents

BDNF Brain-derived neurotrophic factorBG/D Bowman gland and ductBMP Bone morphogenetic proteinFGF2 Fibroblast growth factor 2GAP-43 Growth associated protein of 43-kDa molecular weightGBC Globose basal cellHBC Horizontal basal cellIGF-1 Insulin-like growth factor 1IGSF Immunoglobulin-like superfamilyMeBr Methyl bromideNGF Nerve growth factorNT-3 Neurotrophin 3OMP Olfactory marker proteinOR Odorant receptorOSN Olfactory sensory neuronSus Sustentacular cellPDGF Platelet-derived growth factorTGF-! Transforming growth factor, alphaTGF-" Transforming growth factor, beta

FEATURE ARTICLE THE ANATOMICAL RECORD (NEW ANAT.) 35

by the incorporation of thymidine an-alogues in the normal adult epithe-lium are GBCs (Schwartz Levey et al.,1991; Huard and Schwob, 1995). Bycontrast, HBCs appear more special-ized. The HBCs form hemidesmo-somes with the basal lamina, expresscytokeratins 5 and 14 and a carbohy-drate moiety recognized by Griffonialectin-like basal cells in other epithe-lia, overlie small bundles of axons asthey exit the epithelium, and prolifer-ate at a low rate (Holbrook et al.,1995).

The final cellular element of themucosa is the BG/D unit. The aci-nus, which is composed of cells withabundant secretory granules, resideswithin the lamina propria, and thechannel lined with flattened duct cellsextends to the surface (Farbman,1992). The cells share certain pheno-typic characteristics with Sus cells, in-cluding the expression of identical cy-tokeratins, cytochrome P450s, andanonymous markers recognized bymonoclonal antibodies (MAbs) raisedagainst the epithelium (e.g., MAbsSUS-1 and SUS-4) (Hempstead andMorgan, 1985; Chen et al., 1992;Schwob et al., 1995; Goldstein andSchwob, 1996). As discussed furtherbelow, there is also likely to be a lin-eage relationship between duct cellsand Sus cells, at least during the re-covery of the epithelium after directinjury.

Within many of the categories ofcells, there is an additional diversity oftypes. With respect to the OSNs, di-versity exists at two levels. First, theepithelium can be divided into zonesdefined by the subset of ORs fromwhich the OSNs in that zone can se-lect to express (Ressler et al., 1993;Vassar et al., 1993; Strotmann et al.,1995a; Sullivan et al., 1996), and bythe differential expression of cell-sur-face molecules, for example, the im-munoglobulin superfamily membervariously known as OCAM, RNCAM,NCAM-2 and mamFas II (the lattername intending to indicate the closehomology to Fasciclin II in Drosoph-ila) (Mori et al., 1985; Schwob andGottlieb, 1986; Alenius and Bohm,1997; Paoloni-Giacobino et al., 1997;Yoshihara et al., 1997; Fang, 2001).Second, within a zone of the epithe-lium, neurons differ one from theirneighbor by their selection of the sin-

gle OR they express from among thesubset available (Ressler et al., 1993;Vassar et al., 1993; Strotmann et al.,1995a; Sullivan et al., 1996). Simi-larly, Sus and BG/D cells expressdifferent biotransformation enzymesdepending on epithelial location,as exemplified by differential expres-sion of phenol sulfotransferase G at ahigh level in dorsomedial epithelium(Miyawaki et al., 1996) and the dif-ferential sensitivity of the epithe-lium to injury by assorted olfacto-toxins (Schwob et al., 1994b).

THE PHENOMENA OFOLFACTORY REGENERATIONOne of the singular advantages of thestudy of olfactory regeneration as amodel for general neural developmentis the ease and selectivity with whichthe system can be lesioned in vivo andthe rapidity of its recovery. There aretwo forms of lesion: (1) direct damageto the epithelium by exposure to oneof a variety of toxic agents that dam-age multiple cell types, and (2) selec-tive neuronal degeneration secondaryto axonal damage.

Transection of the OlfactoryNerveSelective loss of neurons can be ac-complished by transection of the largebundles that compose the olfactorynerve as it traverses the cribriformplate. Using a Teflon knife leaves theolfactory bulb mostly intact (althoughsome damage is inevitable with thisprocedure), preserving the potentialfor reinnervation (Costanzo, 1984,1985, 2000; Yee and Costanzo, 1995,1998; Koster and Costanzo, 1996;Christensen et al., 2001). Alterna-tively, the olfactory bulb can be re-moved by aspiration, which both de-stroys the olfactory axons that are incontact with it and eliminates theirsynaptic target (Costanzo and Grazia-dei, 1983; Monti Graziadei, 1983;Schwob et al., 1992). With both ma-nipulations, there is a profound andrapid loss of neurons after their axonsare cut due to retrograde degenera-tion and apoptosis (Monti Graziadeiand Graziadei, 1979; Holcomb et al.,1995). All other cell types are spareddirect damage, including those imma-ture OSNs whose axons have not yetreached the point where the nerve was

lesioned (Fig. 1). In response to thedegeneration, there is a selective andsubstantial increase in the prolifera-tion of GBCs (Schwartz Levey et al.,1991). In the case of olfactory bulbec-tomy, the epithelium never recoversfully even with long survivals after le-sion: there are fewer neurons thannormal, because neuronal lifespan isabbreviated in the absence of trophicsupport from the bulb (Carr and Farb-man, 1992, 1993; Schwob et al., 1992).Moreover, most of the OSNs are im-mature (Monti Graziadei, 1983; Ver-haagen et al., 1990; Schwob et al.,1992). Thus, neurons either do notlive long enough to achieve maturity,or do not persist for long after makingthat transition when born in the ab-sence of their target (Schwob et al.,1992). In the case of nerve transec-tion, the recovery in numbers is morenearly complete, but the rate of GBCproliferation and the percentage ofOSNs that are immature remainhigher than controls (Costanzo, 1984;Christensen et al., 2001). Because ar-eas of the bulb are persistently dener-vated or at best hypoinnervated, theprevalence of immature OSNs is againa likely indication of accelerated pro-duction and death due to the trophicdependence on bulb-derived factors.

Direct Epithelial LesionAgents that are directly olfactotoxicproduce a lesion that is more compli-cated (Fig. 2). Nonetheless, directdamage to the epithelium may bemore analogous to the type of injurythat occurs in a natural setting wherepathogens or toxins are airborne. Theclassic agents, irrigation with ZnSO4

and Triton X-100, kill all cell typesin the affected epithelium (Schultz,1941, 1960; Smith, 1951; Mulvaney,1971; Matulionis, 1975, 1976; Hardinget al., 1978; Burd, 1993). Substantialareas of the mucosa are often spared,presumably due to incomplete or un-even spread of the toxin. In addition,recovery is incomplete, and more se-verely damaged areas that were previ-ously olfactory, reconstitute as respi-ratory epithelium. The inhalation oflow concentrations of methyl bromidegas (MeBr) has proven to be a morereliable and facile means of reversiblydamaging the olfactory epithelium ofrodents (Hurtt et al., 1987, 1988;


Schwob et al., 1994b, 1995). The ol-factotoxic effects of the gas, which arethe sole manifestation of a singlemulti-hour exposure at levels of 200–400 ppm, are probably due to the gen-eration of free radicals by the cyto-chrome P450 system, leading toperoxidative damage to sustentacularcells, neurons, ducts, glands, andbasal cells (Hallier et al., 1994; Yang etal., 1995). Of these, Sus cells andOSNs are completely eliminated fromgreater than 95% of the olfactory epi-thelium, whereas a large percentageof basal cells are spared (Hurtt et al.,1988; Schwob et al., 1995). Despitethe severity of the lesion, there israpid and robust regeneration ofOSNs and Sus cells, such that the ep-ithelium closely resembles unlesionedcontrol by 8 weeks after exposure,e.g., the rate of basal cell proliferation,the ratio of mature to immatureOSNs, cytochrome P450 levels, andother Sus markers have returned tocontrol levels (Hurtt et al., 1988;Schwob et al., 1995).

With both nerve cut and direct epi-thelial lesion, the epithelium also re-covers with respect to its more globalproperties. For example, the divisionof the epithelium into zones definedby differential expression of odorantreceptors and of the IgSF memberOCAM/mamFas II is restored after theepithelium is reconstituted (Schwoband Youngentob, 1992; Schwob et al.,1994b; Christensen et al., 2001). In-deed, the zonal distribution of ORprobe-positive OSNs after recoveryfrom MeBr lesion are indistinguish-able from control when assayed by insitu hybridization for each of 8 differ-ent ORs (Iwema et al., 1997). Zonalityof OR expression is also comparableto control after the partial recoveryfrom the neuron loss caused by bul-bectomy (Konzelmann et al., 1998).Likewise, P2 receptor-expressing OSNsare found in the same epithelial zoneafter recovery from bulb ablation,nerve section, and MeBr exposure(Costanzo, 2000; Iwema and Schwob,2001).

CELLULAR EVENTSACCOMPANYING EPITHELIALREGENERATIONThe foregoing demonstrates the ca-pacity of the olfactory epithelium toreconstitute in two very different set-tings: one where only neurons areneeded to restore the epithelium to itsprelesion condition, and the otherwhere all cellular populations need tobe reconstituted either in whole or inpart. Other self-renewing tissues, forexample, the hematopoietic system,are confronted with a similar task.Thus, there may be a need to recon-stitute whole blood (after hemor-rhage) or particular constituents (forexample, platelets after their selectivedepletion by autoimmune destruc-tion). The capacity for full reconstitu-tion of the blood or other renewabletissues depends on the preservation oftotipotent stem cells, which are ableto give rise ultimately to all types ofcells and to self-renew without appar-ent limitation (Weissman, 2000). At

Figure 2. Cellular constituents and progenitor-progeny relationships in the olfactory epithelium after damage to all categories of cells byexposure to methyl bromide (MeBr) or other form of peripheral olfactotoxin. Distinct stages in the reconstitution of the epithelium occur atdefined time points after the lesion (symbolized by the progressive stages in neuron generation and the thickening of the epithelium movingtoward the right from the center of the figure), allowing them to be studied in relative isolation. Multipotent GBCs are active in giving riseto Sus cells, HBCs, and neurons for at least the first few days after lesion. Neurons do not reappear until 3–4 days after lesion and do notmature until 8–10 days after lesion. HBCs appear reactive in dorsal epithelium. Sus cells also derive from the cells lining the residualBowman’s duct. For abbreviations, see legend to Figure 1.


subsequent stages, the differentiativeand proliferative capacity is progres-sively restricted as progenitor cellspass through a pluri- or multipotentstage to the point where cells are com-mitted to a particular fate either whilecontinuing to divide and expand thepopulation of derivatives (transit am-plifying cells) or producing the finaldifferentiating daughter cells (imme-diate precursor).

The application of a “poietic” modelto the olfactory epithelium has metwith some success. The existing datasuggest that the population of GBCsharbors both transit amplifying cellscommitted to a neuronal lineage, andimmediate neuronal precursors (Figs.1, 2). The pulse-chase experimentswith thymidine analogues mentionedabove first suggested that the popula-tion of GBCs harbors the popula-tion of immediate neuronal precur-sors (Graziadei and Graziadei, 1979;Schwartz Levey et al., 1991). Resultsfrom studies of neurogenesis in vitroalso indicate that some GBCs are im-mediate neuronal precursors (Calofand Chikaraishi, 1989). Finally, themost direct evidence comes from theuse of replication-incompetent, retro-virally derived vectors to define lin-eage (Caggiano et al., 1994; Schwob etal., 1994a). In these experiments, vec-tor-labeled OSNs are observed in ex-clusive association with GBCs afterdirect injection of the vector into theepithelium. Rarely, HBC-only clustersare observed (Caggiano et al., 1994).The timing and spatial pattern ofexpression of the basic helix-loop-he-lix transcription factor neurogenin-1during development suggests that it isexpressed by those GBCs that func-tion as immediate neuronal precur-sors (Cau et al., 1997). In addition,another subset of GBCs, which ex-press the transcription factor MASH1,seem to be transit amplifying cells(Guillemot and Joyner, 1993; Gordonet al., 1995). That functional assign-ment is based on the expansion of theMASH1 (#) population in advance ofthe increase in the overall rate of GBCproliferation and upstream of the neu-ronal differentiation factor neuroge-nin1 (Gordon et al., 1995; Cau et al.,1997). The latter interpretation fitswith the absence of neurons from theolfactory epithelium in Mash1 knock-out mice (Guillemot et al., 1993).

The foregoing data, derived fromexperimental settings in which onlyneurons are being generated at a sub-stantial rate, have been interpretedfrom a somewhat neurocentric view-point as indicating that GBCs serveonly as committed neuronal progeni-tors (Calof et al., 1998). According tothis view, any need to replace Suscells, HBCs, or BG/D is satisfied byself-renewal wherein existing Sus cellswould generate new Sus cells, etc. Astem cell capable of continuing to re-new the neuronal population has ei-ther been localized with GBCs or withthe population of HBCs (Mackay-Simand Kittel, 1991; Mumm et al., 1996);the assignment of stem cell functionto HBCs has been based, in large part,on the identification of cells that areintermediate in appearance betweenHBCs and GBCs (Graziadei andMonti Graziadei, 1978; Graziadei andGraziadei, 1979).

A slightly more complex view ofprogenitor cell capacity emerges fromthe detailed study of the recovery ofthe epithelium after MeBr lesion (Fig.2). In this setting, three different strat-egies—lineage tracing, tissue analysisby using cell-specific markers, andtransplantation of marker-selectedcell types into the epithelium—all sug-gest that at least some GBCs andBG/D cells have a broader differentia-tive capacity than that observed innormal or bulbectomized epithelium.The application of lineage-tracing vec-tors to the MeBr-lesioned epitheliumis facilitated by the fact that the pro-liferating cells can be infected bymeans of simple infusion into the na-sal cavity after destruction of neuronsand sustentacular cells (Huard et al.,1998). In addition, diluting the vectorthroughout the nasal cavity ensuresthat only a single progenitor cell isinfected productively within a partic-ular area of the epithelium. As a con-sequence, clusters of vector-labeledcells are clonal in this paradigm.Quite strikingly, the data indicate thatsingle progenitor cells give rise to bothneurons and non-neuronal cells, in-cluding Sus cells, microvillar cells,and HBCs, in addition to GBCs andneurons. Other clones are composedof Sus cells plus BG/D cells. Finally,some clones encompass only neuronsand GBCs, or only Sus cells. The latterdata are expected given the kinetic

and lineage tracing data in normal orbulbectomized epithelium referencedabove and indicate that some progen-itor cells are fated to give rise to asingle lineage even in the setting of theMeBr-lesioned epithelium. Neuronsand GBCs are never observed in thesame clone as BG/D cells.

Analysis of marker expression inMeBr-lesioned tissue is also consis-tent with the lineage results (Gold-stein and Schwob, 1996). Antibodiesthat label GBCs and neurons in thenormal epithelium are coexpressed oncells with HBC or Sus cell character-istics during the first few days afterMeBr lesion. The most parsimoniousinterpretation of the data is that bothHBCs and Sus cells can be generatedby GBCs in a setting in which all celltypes are being reconstituted. That is,the immunohistochemical analysisalso suggests that some GBCs aremultipotent in their differentiative ca-pacity. In addition, Sus and BG/D-spe-cific antibodies label cells that look tobe crawling away from the ductsalong the surface of the epitheliumand are likely to be the source of thelineage association between Sus cellsand BG/D cells noted above (Schwobet al., 1995).

Finally, transplantation of labeledcells also confirms the interpretationthat GBCs are multipotent in their ca-pacity (Goldstein et al., 1998). In oneset of experiments cells were dissoci-ated and harvested from the OE ofanimals shortly after bulbectomy, la-beled ex vivo by infection with a con-ventional retroviral vector or with ahighly complex library of vectors(with a theoretical upper limit of 107

sequence-distinct vectors), and thentransplanted into the OE of MeBr-le-sioned host rats. The high numeri-cal complexity of the vector libraryensures that each tag representsa unique infective event, allowingclonality of descendents to be proved.The vast majority of cells that are in-fectible by the vector, i.e., the vast ma-jority of actively proliferating cells inthe bulbectomized epithelium, areGBCs. Clones that include both neu-rons and the full range of non-neuro-nal cells are observed as was de-scribed above for the lineage-tracingexperiments. These data have two im-plications. First, some GBCs are de-monstrably multipotent in this assay.


Second, some of the GBCs that areactively proliferating in a setting inwhich only neurons are being re-placed are not irreversibly committedto generate neurons alone (Fig. 1).Rather, the data suggest that some ofthe GBCs in a purely neurogenic epi-thelium are merely fated to make neu-rons and are not irreversibly commit-ted to do so. Thus, the decision tomake only neurons versus both neu-rons and non-neuronal cells in neuro-genic epithelium is one that is madecontinually by actively proliferatingprogenitors. In another set of experi-ments, GBCs were selectively isolatedfrom bulbectomized epithelium by us-ing a combination of antibody mark-ers and fluorescence-activated cellsorting (FACS) (Chen et al., 2001). Af-ter infection with the vector, the GBCpreparation is transplanted into theMeBr-lesioned host epithelium. Theresults are preliminary, but the find-ing that neurons and Sus cells derivefrom GBCs in the context of theMeBr-lesioned and recovering epithe-lium support strongly the interpreta-tion that at least some GBCs are mul-tipotent.

MOLECULAR REGULATION OFOLFACTORY EPITHELIOPOIESISThe cellular events described aboveindicate that the progenitor cell pop-ulation in the olfactory epithelium issubject to regulation at multiple lev-els. For example, neurogenesis accel-erates when a wave of dying OSNs,killed by nerve transection, imposesan increased demand for replace-ment neurons (Schwartz Levey et al.,1991; Carr and Farbman, 1992, 1993;Schwob et al., 1992). Upstream of acommitment to the selective produc-tion of neurons, some GBCs are capa-ble of giving rise to both neurons andnon-neuronal cells and do so in thecontext of an epithelium in which allcell types have been depleted by le-sion, as reviewed above. The molecu-lar regulation of the aforementionedcellular events is incompletely under-stood (Fig. 3).

What we know has come from threeapproaches: primary cell culture (bothdissociated and explant), olfactory-de-rived cell lines, and transgenic knock-out mice. A number of agents increaseproliferation of the various populations

of epithelial cells, including FGF2,EGF, TGF-!, and PDGF, from amongthe usual suspects (Calof et al., 1991;Mahanthappa and Schwarting, 1993;Farbman and Buchholz, 1996; Gold-stein et al., 1997). Remarkably, OMPalso stimulates cell division in explantcultures (Farbman and Ezeh, 2000).Other factors have been shown to pro-mote differentiation and suppress fur-ther proliferation, including TGF-"sand BMPs (Mahanthappa and Schwart-ing, 1993; Shou et al., 1999, 2000). Fi-nally, the neurotrophins NGF, BDNF,and NT-3, or other factors such asIGF-1, may also be crucial for survivaland differentiation (Roskams et al.,1996; Pixley et al., 1998). For none ofthese has the analysis been completeenough to allow us to assert their roleunequivocally in the overall cellulareconomy of the epithelium in vivo.

Nonetheless, there is sufficient consen-sus among the published data to justifytheir examination.

The Neurogenic CascadeMackay-Sim, Chuah, and colleagueshave proposed a sequential model forthe molecular regulation of neurogen-esis (Mackay-Sim and Chuah, 2000;Newman et al., 2000). Even though notall features of this model are equallywell-established, it is a heuristically use-ful framework for the consideration ofthe data regarding growth factor con-trol (Fig. 3). In their view, a lineage re-lationship between HBCs and OSNs re-mains uncertain but possible. Indeed,there are no direct data in favor of thenotion that HBCs are the epithelialstem cells responsible for maintainingneurogenesis in the long term, and

some evidence against it (Holbrook etal., 1995). HBCs in semi- or fully disso-ciated cultures, in explants, and in vivoproliferate in response to growth fac-tors that activate the ErbB family ofreceptors, including TGF-! and EGF(Mahanthappa and Schwarting, 1993;Farbman and Buchholz, 1996; Ezehand Farbman, 1998; Farbman andEzeh, 2000; Getchell et al., 2000). Thereis evidence that HBCs express the EGFreceptor (ErbB-1) (Holbrook et al.,1995; Krishna et al., 1996) and the neureceptor (ErbB-2) (Salehi-Ashtiani andFarbman, 1996; Ezeh and Farbman,1998). TGF-! is the relevant ligand invivo; basal cells (not otherwise specifiedas to type), sustentacular cells, and BGsstain with anti–TGF-! antibodies,whereas EGF has not been demon-strated in the mucosa (Farbman andBuchholz, 1996). In keeping with thedata in vitro, insertion of a CK14 pro-moter-TGF-! transgene to drive overex-pression of the growth factor in HBCscauses a multifold, selective increase inproliferation of the HBCs, presumablyby means of a paracrine pathway(Getchell et al., 2000). TGF-! is unlikelyto be the sole element regulating HBCproliferation; the elimination of TGF-!by genetic recombination does notabolish the already low level of prolifer-ation observed in otherwise normal OE(Getchell et al., 2000). Assaying HBCproliferation after MeBr exposure, be-cause HBCs proliferate in this “natural”setting (Schwob et al., 1995), may pro-vide a more sensitive measure for dem-onstrating whether TGF-! is an impor-tant regulator of HBC behavior in vivo.

A bit more is known about the con-trols on GBC proliferation and differ-entiation. Growth factor applicationto semidissociated and dissociatedOE cells in culture demonstrates thatFGF2 is a capable of stimulating theproliferation of GBCs (Calof et al.,1991; DeHamer et al., 1994; Mumm etal., 1996; Newman et al., 2000). Mostof the cells divide only once or twicein the presence of FGF2, generating asmall number of neurons in vitro, i.e.,FGF2 appears to drive immediateneuronal precursors. A small percent-age of GBCs may respond to FGF2 byundergoing a more pronounced pro-liferative response, which leads to theformation of larger colonies of neu-rons (DeHamer et al., 1994). Someslight acceleration of proliferation by

The decision to makeonly neurons versus both

neurons and non-neuronal cells in

regeneratingneurogenic epithelium is

made continually byactively proliferating

progenitors.


FGF2 is also seen with olfactory-de-rived, spontaneously immortalizedcell lines, but to a lesser degree than isobserved with a richer medium con-taining serum and tissue extracts(Goldstein et al., 1997; Ensoli et al.,1998). For the cell lines, the morestriking effect is the suppression ofneuronal differentiation by FGF2(Goldstein et al., 1997). Taken to-gether, the data from both suggestthat FGF2 may be preparing GBCs torespond to other signals rather thanserving as potent mitogen per se. Thathypothesis is consistent with the ac-tion of FGFs in other differentiatingtissues, including muscle (Lathrop etal., 1985; Itoh et al., 1996). Analysis invivo has been limited to localization ofFGF2 and members of the family of

FGF-Rs in the OE. FGF2 is found inneurons and sustentacular cells inparts of the epithelium (Goldstein etal., 1997). Members of the FGF-Rfamily that respond to FGF2 have alsobeen found in OE by RT-PCR (De-Hamer et al., 1994).

Forms of Negative Feedbackon NeurogenesisNegative regulators of neurogenesishave also been tentatively identified.This line of investigation derives fromin vivo phenomena: GBC proliferationaccelerates when OSN death is en-hanced by nerve transection or bulbablation, but slows in settings wherethe neurons are protected and proba-bly longer-lived, i.e., when one side of

the nasal cavity is isolated from theenvironment by naris closure (Farb-man, 1990). A more direct demonstra-tion of negative feedback on neuro-genesis is the suppression of neuronalcolony formation in vitro when cellsare cultured with a large excess of dif-ferentiated neurons (Mumm et al.,1996). The anti-neurogenic effects ofadding neurons to the cultures aremimicked by exogenous BMPs 2, 4,and 7 (Shou et al., 1999, 2000); BMP7apparently exerts a bimodal effect,stimulating neuronal survival at lowconcentrations and suppressing neu-rogenesis at higher levels (Shou et al.,2000). In this setting, BMPs cause therapid degradation of MASH1 protein(Shou et al., 1999, 2000). It seems thatthe MASH1-expressing cells die invitro after the BMP-induced degrada-tion of MASH1 as they do in vivo inanimals in which the Mash1 gene hasbeen disrupted by homologous re-combination (Shou et al., 2000). TheBMPs are expressed in nasal tissue invivo; BMPs 4 and 7 are expressed infetal OE, although cellular localiza-tion is incomplete, and all three arefound in fibroblasts of the underlyingstroma (Shou et al., 2000).

Other members of the TGF-" super-family, specifically TGF-"1 and TGF-"2, promote neuronal differentiationin semidissociated and dissociatedprimary cultures of OE and in OE-derived cell lines (Mahanthappa andSchwarting, 1993; Newman et al.,2000). In all of these settings, the ex-pression of neuronal markers, includ-ing neuron-specific "-tubulin andNCAM, is markedly up-regulated. Inmany other tissues, TGF superfamilymembers have been shown to act sim-ilarly. The heterodimer PDGF-AB iseffective at preserving the differentiat-ing cells for at least a brief period oftime and within a narrow range ofconcentration (Newman, 2000). Otherfactors that are expressed in the OE,including BDNF, were ineffective atmaintaining long-term neuronal sur-vival in vitro (Holcomb et al., 1995).Indeed, elimination of BDNF, NT-3,or both, by single and double geneknockout does not cause any disrup-tion of OE structure or axon targetingduring embryonic development (Nefet al., 2001).

Figure 3. Growth factor regulation on the processes of cellular renewal in the olfactoryepithelium apparently depends on similar factors playing similar roles as in other systems.The balance between fibroblast growth factor-2 (FGF-2) and BMP signals seems to becritical for advancing or retarding neurogenesis. Transforming growth factors-" (TGF-"s)stimulate neuronal differentiation in cell lines derived from GBCs. Lines ending in circlesdesignate a stimulatory effect; lines ending in a perpendicular line indicate inhibition. IGF-1,insulin-like growth factor-1; PDGF, platelet-derived growth factor. For other abbreviations,see legend to Figure 1.


LIMITS ON THE REINNERVATIONOF THE OLFACTORY BULB AFTERLESIONThe capacity for reconstitution of theneuronal population, whether partialor complete, raises the obvious issuesof how the olfactory system accom-plishes the re-innervation of the adultCNS (not true of other neural sys-tems) and the specificity with whichthe bulb is reinnervated during theregeneration process. It seems certainthat the glial cell environment of theolfactory nerve and the superficial lay-ers of the olfactory bulb is fundamen-tal to the robustness of regeneration.The relationship between axons andensheathing cells of the olfactorynerve and olfactory glomerular layer,derived from the olfactory placode, ishighly reminiscent of that between pe-ripheral axons and Schwann cells atan early stage in their embryonic de-

velopment (Bunge, 1968; Farbman,1992). In both settings, the glial cellelement ensheathes multiple axonsand holds them in juxtaposition, butin the case of the olfactory nerve, eachenwrapped fascicle is composed ofhundreds of axons (Farbman, 1992).The close apposition of axon to axonlikely provides a highly favorable sub-strate for subsequent growth. The dis-tinct segregation and aggregation ofolfactory axons is maintained into theglomerulus, in which central glia areapparently restricted to the periphery(Raisman, 1985; Kasowski et al.,1999). The robustness of olfactorygrowth is reproduced in part when ol-factory glia are transplanted into thelesioned spinal cord fostering both an-atomical and functional recovery, orin the dorsal root entry zone promot-ing the growth of damaged dorsal rootfibers back into the CNS (Ramon-

Cueto and Nieto-Sampedro, 1994; Liet al., 1997; Raisman, 2001). The mo-lecular features of the olfactory gliaresponsible for their axonal growthpromoting properties remain un-known.

Having reached the bulb, newlyinnervating axons need to form spe-cific connections within the glomer-ular layer. The connectivity of ol-factory axons with the bulb seems tobe specified at a minimum of threelevels (Fig. 4). At the broadest level,the subdivision of the epitheliuminto zones, defined by the pattern ofOR expression, is maintained in theprojection of olfactory axons ontothe bulb, establishing a rhinotopicarrangement between sensory pe-riphery and central target, whichmay be important for translatingthe physicochemical interaction ofodorant and nasal space (Moulton,

Figure 4. Glomerular targeting during neuronal turnover at a low level. In the primary olfactory projection of “normal” animals, or oneswhere a substantial population of preexisting neurons and axons survive lesion, newly born neurons are confronted with an environmentin which axons that are alike in terms of the odorant receptor (OR) that is expressed can serve to guide later-arriving like-axons (indicatedby the symbolic growth cone at their tips). In this setting, glomerular convergence is seemingly preserved throughout life. OSN, olfactorysensory neuron.


1976; Schoenfeld et al., 1994; Key-hani et al., 1997). High level expres-sion of the Ig superfamily memberOCAM/mamFas II is limited to theventrolateral zones, allowing projec-tions from dorsomedial and ventro-lateral OE to be distinguished onthat basis, or by means of more con-ventional retrograde tract tracing ex-periments (Mori et al., 1985; Schwoband Gottlieb, 1986; Yoshihara et al.,1997). At its most precise, the con-vergence of axons elaborated byOSNs that express the same OR ontoone or a few glomeruli on the medialand lateral faces of the bulb estab-lishes a kind of receptotopy that de-pends in some manner on the selec-tion of the OR to be expressed(Ressler et al., 1994; Vassar et al.,1994; Mombaerts et al., 1996; Wanget al., 1998). However, the locationof an OR’s “target glomerulus” isspecified only to the extent that itfalls within an array of glomeruliroughly 30 –50 in number whencompared among conspecifics(Strotmann et al., 2000; Schaefer etal., 2001). Thus, there is an interme-diate level of specificity, i.e., target-ing to a subarray of contiguous glo-meruli, before the final event ofglomerular acquisition.

Anatomical and FunctionalConstancy in the AdultIs connectivity in the adult main-tained with a specificity equivalent tothat established during embryonic de-velopment? It appears that specificconnectivity is maintained at the lowrate of turnover that occurs duringadult life in a protected laboratory set-ting, although the issue has been sub-ject to only limited examination (Fig.4). For example, in aged animals,the glomerular innervation by P2axons in P2-IRES-tauLacZ mice isroughly equivalent to weanling ani-mals (Costanzo, personal communi-cation; Schwob, unpublished results).The labeled fibers converge and ap-parently do not wander into aberrantglomeruli, although the incidence ofmultiple glomeruli at a single bulbarsite may be increased. Anatomicalconstancy correlates well with theusual human experience of perceptualstability throughout life. However, thewholesale replacement of the neuro-

nal population that is elicited by ei-ther nerve transection or MeBr lesionis a different task and may be infor-mative regarding the intrinsic capaci-ties for accurate targeting in the adult.

Reinnervation: Nerve Damagevs. Epithelial LesionThe two paradigms, nerve damageversus epithelial lesion, differ with re-spect to the condition of the olfactorynerve. With transection, the fascicularstructure and blood supply of thenerve is markedly disrupted (Berger,1971a,b; Graziadei, 1974; Schwob etal., 1994b), which leads to invasion byblood-borne macrophages, judgingfrom events in other traumaticallydamaged peripheral nerves (Berger,1971a; Taskinen and Roytta, 1997).With MeBr exposure or other periph-

eral agent, the nerve is left largely un-affected by the damage to the OE, al-though there is some glial reaction tothe massive wave of axonal degenera-tion (Schwob et al., 1999). Reinnerva-tion of the bulb occurs rapidly in bothsettings. By the second-to-third weekafter either form of lesion, axons havere-entered most areas of the olfac-tory nerve layer and have growninto underlying glomeruli (Graziadeiet al., 1980; Yee and Costanzo, 1995;Schwob et al., 1999; Costanzo, 2000;Christensen et al., 2001).

In the case of olfactory nerve tran-section, significant errors occur dur-ing the reinnervation of the bulb at alllevels at which the projection is orga-nized: rhinotopy, selection of glomer-ular subarray, and receptotopic glo-merular convergence. Areas of thedorsomedial bulb remain perma-

nently denervated or profoundly hy-poinnervated, whereas axons from thedorsomedial OE that ought to projectthere are misdirected into territoryusually occupied by fibers from theventrolateral OE (Christensen et al.,2001). Furthermore, the projection ofOR-defined subsets of OSNs is not re-stored to its prelesion state. For exam-ple, P2 fibers converge onto glomerulibut do not target the same area of thebulb as in normal P2 mice, and typi-cally innervate multiple, widely dis-persed glomeruli (Costanzo, 2000).

In the case of MeBr-mediated de-struction of the OE, the zonal/rhino-topic organization of projection ap-proaches prelesion in the cases forwhich the degree of damage permitsfull or nearly full recovery of the epi-thelium (Fig. 5) (Schwob and Young-entob, 1992). In these experiments,the projection was assessed both byimmunostaining by using the anti-OCAM/mamFas II MAb designatedRB-8 and by retrograde transport offluorescently labeled microspheres.However, if the lesion is more severe,which causes some areas of the ante-rior, ventral, and lateral OE to un-dergo respiratory metaplasia (i.e., thepatchy replacement of olfactory by re-spiratory epithelium during recoveryafter injury), then the posterior mar-gin of the bulb remains denervated,indicating that the normal projectionto those glomeruli has been redirectedto more anterior glomeruli (Schwobet al., 1999).

Failure of Glomerular TargetingThe failure to reinnervate the poste-rior bulb seems to be the most strikingmanifestation of a general failure inchoosing precisely the original glo-merular subarray for reinnervationand in the convergence of like axons,both of which normally maintain thesharply focused receptotopic pattern-ing of the projection (Fig. 5). That in-terpretation is confirmed by demon-strations of mistargeting of neuronalsubsets defined by OR expression (i.e.,assaying P2 fibers in mice after MeBrlesion) (Iwema and Schwob, 2001) orby expression of other molecularmarkers that apparently correlatewith OR expression, for example MAb2A4, which labels neurons projectingto a single pair of glomeruli in the

It seems certain that theglial cell environment ofthe olfactory nerve andthe superficial layers of

the olfactory bulb isfundamental to the

robustness ofregeneration.


normal rat (Carr et al., 1994, 1998),and MAbs 213 and 2C6, which labeldistinct sets of neurons projecting tolargely nonoverlapping groups ofnecklace glomeruli (Ring et al., 1995,1997). In these examples, it appearsthat at least some axons approach thecorrect glomerular subarray, but thetight convergence onto one or a fewgrouped glomeruli is seriously dis-torted (Ring et al., 1995; Carr et al.,1998). Similarly, a return of axons toroughly the right area of the glomerularlayer is observed with larger groupingsof identified neurons after Triton X-ir-rigation of the nose of H-OMP-LacZ-6transgenic mice (Cummings et al.,2000).

The example of the necklace glo-meruli is particularly informative, asthose glomeruli can be identified from

animal to animal solely on the basis ofposition and structure in conventionalhistological sections (Ring et al.,1997). In cases for which the lesion issevere, the staining with MAb 2C6 of-fers a clear demonstration that fibersthat would normally target the identi-fiable glomeruli in the posterior bulbend up in more anterior regions, i.e.,the fibers approach the usual targetarea, defined broadly as the set of glo-meruli adjacent or near to the normalnecklace, but fall short and do notconverge onto a bona fide necklaceglomerulus (Ring et al., 1995). In con-trast, in animals for which the lesionwas less severe, the appropriate neck-lace glomerulus is reinnervated (Ringet al., 1995). The difference seems toreflect whether a large enough popu-lation of neurons is spared, as is the

case with the less severe lesion, to of-fer guidance at the final, glomerularacquisition stage of the reinnervationprocess. In the absence of the preex-isting fibers, as is the case with themore severe lesion, the reinnervatingaxons do not acquire their prelesionglomerulus.

The selective denervation at theposterior margin of the bulb when thepopulation of innervating neurons isreduced in magnitude due to respira-tory metaplasia suggests that a ten-dency for newly arriving fibers to fillpreferentially glomeruli that are closeto the cribriform plate and the initialpoints of contact between olfactoryaxons and the bulb may play a signif-icant role in the patterning of reinner-vation (Schwob et al., 1999). The ap-parent tendency to fill available

Figure 5. Glomerular targeting following wholesale neuronal turnover. In contrast to the normal setting, wholesale turnover of the neuronalpopulation eliminates the possibility of using preexisting like-fibers as guides for the final stage of glomerular targeting. In the case ofMeBr-lesioned animals, as illustrated here, rhinotopy is restored, such that high OCAM/mamFas II-expressing olfactory sensory neurons(OSNs) reinnervate the same area of the bulb, and low OCAM/mamFas II-expressing OSNs reinnervate the complementary area of thebulb, as in normal. In addition, odorant receptor (OR) -defined subsets of neurons return to roughly the same subarray of the glomerularlayer of the bulb (consisting of probably 30–50 glomeruli) as in normal. However, the final stage of glomerular convergence is notaccomplished, and axons distribute to multiple glomeruli within the subarray. Thus, the mechanisms that achieve targeting to the level ofthe subarray seem to be maintained throughout adulthood.


glomerular space may be an impor-tant feature of the reinnervation of thebulb, and may differ from the devel-oping projection. The preference foran unoccupied target may also featurein the restoration of the precise prele-sion pattern after targeted ablation ofa single OR-defined subset of olfac-tory neurons by means of applicationof transgenic technology.

In an elegant experiment from Ax-el’s group, the neurons that expressOR P2 are killed by the specific, buttransient coexpression of diphtheriatoxin in concert with the P2 receptor(Gogos et al., 2000). Replacement P2neurons are regenerated, at least inpart, over a time course of a month(without any preferential productionof P2 neurons, apparently). Their ax-ons converge onto glomeruli that arein analogous positions as their wild-type counterparts. The re-emergenceof OR-specific targeting is impressiveand has been interpreted as an indica-tion that the adult system retains theinformation needed to restore bothzonality and receptotopy (Gogos et al.,2000). However, only the P2 specificglomeruli are denervated. To the ex-tent that reinnervating P2 fibers ap-proach their usual glomerular subar-ray under the control of otherguidance mechanisms and then pref-erentially occupy a vacated glomeru-lus, the paradigm may not be indica-tive of receptotopic accuracy, per se.

Consequences of AlteredConnectivityHow does the inaccuracy in axon tar-geting impact on behavioral recoveryafter injury? Surprisingly little, giventhe shifting and blurring of the recep-totopic map onto the bulb suggestedby the existing, albeit limited, analysisin nerve transected and MeBr-le-sioned animals. For example, anatom-ical recovery after knife cut of the ol-factory nerve is sufficient to allow thelesioned animal to make simple odor-ant discriminations (Yee and Cos-tanzo, 1995, 1998). Similarly, MeBr-lesioned and recovered animals re-tain a fairly complex odorant identifi-cation task learned before expos-ure (Youngentob and Schwob, 1997;Schwob and Youngentob, 2001). Inthis case, the animals had beentrained to associate each of five oper-

ant tunnels with a distinct odorantand had learned that task to greaterthan 90% accuracy; the nature of theanalysis allows one to construct a 5 $5 matrix of odorant stimuli and re-sponse alternatives. Lesioned animalsrested without testing for 2 monthsafter lesion—a time period sufficientto allow anatomical recovery—re-quire a few more days of testing toreturn to precriterion levels of perfor-mance with respect to percentage cor-rect than control rats merely restedfor 2 months without lesion. Nonethe-less, the previously lesioned animalsdo recover to a remarkable degree.

Additional insight into the neuralprocessing of the odorant stimuli canbe gleaned from analysis of the pat-tern of responses across the matrix,both on-diagonal correct ones and off-diagonal errors, by means of multidi-

mensional scaling analysis (Youngen-tob et al., 1991, 2001; Youngentob,2001). Indeed, application of multidi-mensional scaling analysis does reveala subtle alteration in encoding of ol-factory stimuli in the normal vs. re-generated system. When the twogroups of rats were compared on thebasis of the pattern of responses, thecontrol animals were found to clustertightly together in the multidimen-sional space in which the patterns arelocated, whereas the lesioned and re-covered animals are at a further re-move from the controls and from eachother. The analysis indicates that le-sioned/recovered animals are process-ing stimuli in a different manner fromcontrols. The conjunction of the ana-tomical and behavioral results indi-cates that the spatial mapping ofodorant representation across the glo-

merular surface is important for theperception of odorant stimuli.

CONCLUSIONS, UNRESOLVEDISSUES, POTENTIAL STRATEGIESI have tried to present a broad view ofcellular renewal in the olfactory epi-thelium, emphasizing the need to sus-tain both neuronal and non-neuronalcell populations in the face of continu-ing environmental assaults to main-tain sensory function. Given the usualform of damage to which the epithe-lium is subject, analyses limited toneurogenesis are insufficient to un-derstand the biology of the system.Making experimental lesions thatdamage other cell types in the epithe-lium as well as neurons reveals unan-ticipated capacities. The data re-viewed here show that the adultepithelium retains cells that are mul-tipotent in their differentiative capac-ity. Furthermore, cells with the capac-ity to give rise to nearly all epithelialcell types—OSNs, Sus cells, HBCs,and GBCs—are likely to reside withinthe broad category of GBCs. Lineagetracing analyses, cell marker studies,and transplantation experiments allsupport the assignment of multipo-tency to some type of GBC. That themultipotent stem cells encapsulate acritical capacity is shown by the asso-ciation between destruction of GBCsand the initiation of respiratory meta-plasia after severe, direct injury to theepithelium (Schwob et al., 1995). Inaddition, the apparent capacity of BDcells to give rise to Sus cells indicatesthat Sus cells can arise by means oftwo distinct lineages, which is un-usual (Huard et al., 1998).

Unfortunately, we are currently un-able to identify which subset of GBCsact as multipotent progenitors. MASH1(#) GBCs seem to act as transit am-plifying cells committed to the neuro-nal lineage, whereas those expressingneurogenenin1 seem to function asimmediate neuronal precursors basedon current data (Gordon et al., 1995;Cau et al., 1997). Molecularly anony-mous antibody markers, including theones that we developed (Goldsteinand Schwob, 1996; Goldstein et al.,1997; Jang and Schwob, 2001) or an-tibodies to other cell differentiationantigens, may provide an alternativemeans of partitioning the broader

Lineage tracinganalyses, cell marker

studies, andtransplantation

experiments all supportthe assignment of

multipotency to sometype of globose

basal cell.


population of GBCs into functionallydefined subsets. Fundamental to anyattempt to define functional capacityis the nature of the assay to be used,because the definition of commitment,i.e., distinguishing between differentia-tive fate vs. differentiative capacity, is anoperational one. Transplantation ofmarked cells into the epithelium of aMeBr-lesioned animal has the poten-tial for offering the donor cells a set-ting where the all types of cells arebeing generated at a robust rate, i.e.,all progenitors can be and are beingactivated by the signals needed to di-rect the full range of differentiativepotential. The olfactory transplanta-tion assay is intended to mimic thecolony forming unit assay-spleen (oflethally irradiated mice) that revolu-tionized the study of hematopoiesis,with the added advantage that thehost environment is responding to anevent that is highly analogous to ordi-nary life.

The model for epitheliopoiesis pre-sented here differs from the more se-lectively neurogenic models of otherinvestigators in the field. For example,in one type of assay partially purifieddissociated OE cells are placed ontostromal substrates (Mumm et al.,1996). The suppression of neurogen-esis by the addition of exogenous neu-rons or BMPs was referenced above.Although the emphasis was on colo-nies composed largely of neurons,other colonies were composed of cellswith “non-neuronal” phenotypes, in-cluding ones that were “epithelial” inappearance. The epithelioid cells donot look like neurons in vivo, nor dothey resemble the neurons of simplespindle morphology that have beenidentified in semidissociated cultures.However, they do resemble ones thatexpress GBC antigens and low levelsof neurotubulin and NCAM proteinand can be pushed to make the neu-ronal antigens at higher levels andsuppress the GBC antigens by the ad-dition of TGF-"s (Newman et al.,2000). The failure to achieve full neu-ronal differentiation may reflect theabsence of signals in the dissociatedcultures that are needed to promotewhat may be less committed GBCs.

Likewise, the capacity of GBCs togive rise to non-neuronal cells mayrequire cell–cell contacts or other sig-nals, as yet unidentified, that establish

a competency state that permits theGBCs to differentiate along a non-neuronal pathway. According to thisformulation, neurogenesis may be thedefault competency of some or allGBCs. However, that component ofthe GBC population situated up-stream of the transit amplifying cell,should they be limited to making neu-rons under the culture conditions gen-erally in use, is not irreversibly com-mitted to that fate (Goldstein et al.,1998; Huard et al., 1998). The data arenot inconsistent with the hypothesisthat a broader-than-neuronal capacityhas to be elicited in some GBCs by apositive signal in contrast to the alter-native, whereby multipotency is ac-tively suppressed and the absence ofthe purported negative cue is suffi-

cient to release their capacity to makenon-neuronal cells.

The foregoing hypothesis may bemore consonant with the behavior ofother kinds of stem cells. For example,hematopoietic stem cells have the ca-pacity to give rise to other than bloodcells, e.g., neurons, when in the corre-sponding environment, e.g., the brain(Anderson et al., 2001). Clarity will beachieved in the context of the olfac-tory system only when we are betterable to subdivide the population ofGBCs and assess their differentiativecapacity.

Even when that strategy is fully suc-cessful, we will still be left with thedifficulties of assessing the cellularand molecular cues that regulate epi-thelial renewal during normal life and

after injury. The standard manipula-tions—adding growth factors, block-ing transduction cascades, knocking-out genes—will all help to clarify theregulatory networks. We may have anadvantage in the olfactory system overother neural systems by virtue of theease with which cells can be put backinto the epithelium after lesion (Gold-stein et al., 1998). In particular, wehave recent preliminary results sug-gesting that conditionally immortal-ized cells can be stably integrated intohost epithelium after being returnedto the animal (Chen et al., 2001). If theresults are substantiated, the avail-ability of transplantable cell linessuggests an alternative approach.Namely, it will likely be feasible tomanipulate the genome of condition-ally immortalized cells in vitro andthen assess the consequences for theirdifferentiation after transplantation.In another potentially powerful ap-proach, embryonic stem cells mightbe manipulated, induced, and thentransplanted. By using either of theseapproaches, we will be able to definethe receptive side of the relevant sig-naling pathways with great facility.

Beyond the question of how andhow well the epithelium recovers afterinjury remains the issue of the mech-anisms underlying the accuracy withwhich the bulb is reinnervated andhow errors arise. That a hierarchy ofmechanisms culminates in the recep-totopic map across the olfactory glo-meruli remains the most likely expla-nation for the establishment of themap during development, its mainte-nance in adulthood, and its restora-tion after lesion (to the extent thatrecovery occurs). The molecular regu-lation of those events leading up to thefinal stage of glomerular innervationremains obscure. Candidate mole-cules and processes include sema-phorins, ephrins, and various glyco-protein moieties (Treloar et al., 1996;Whitesides and LaMantia, 1996; Dow-sing et al., 1997; Kafitz and Greer,1997, 1998a; Raabe et al., 1997; Tenne-Brown et al., 1998; Tisay and Key,1999; Crandall et al., 2000; Schwart-ing et al., 2000; St John et al., 2000; StJohn and Key, 2001). Molecular ge-netic techniques suggest that the ORthat is expressed plays a role in thefinal acquisition of the glomerular tar-get (Singer et al., 1995; Mombaerts et

The goal ofunderstanding how and

why regeneration —including reconstitutionof the epithelium and

reinnervation of the bulb— remains facile in this

part of the nervoussystem and not others is,

if not within reach, atleast within view.


al., 1996; Wang et al., 1998). It is likelythat the special properties associatedwith the glia of the olfactory nerve andolfactory nerve layer play a major rolein supporting growing axons and mayalso be crucial in guiding them bysome as yet undetermined means(Kafitz and Greer, 1998b, 1999; Bar-tolomei and Greer, 2000). The rein-nervation of the bulb initiated byMeBr lesion may be an ideal in vivomodel for most levels of the axon tar-geting process, because axons returnto the appropriate area of the bulb,even though the final stage—i.e., glo-merular acquisition—is not accom-plished in the absence of preexistingfibers.

Despite the current limitations onour understanding, much has beenlearned over the recent past about theevents and the regulation of the regen-eration of the primary olfactory sys-tem after injury. The armamentariumthat has been and continues to be as-sembled promises even more rapidadvances in the future. The goal ofunderstanding how and why regener-ation—including reconstitution of theepithelium and reinnervation of thebulb—remains facile in this part ofthe nervous system and not others is,if not within reach, at least withinview.

ACKNOWLEDGMENTSI thank the members, past andpresent, of my lab for their tirelesscontributions in support of this work,in particular Xueyan Chen, Heng-sheng Fang, Bradley Goldstein, JohnHamlin, Eric Holbrook, Josee Huard,Carrie Iwema, Woochan Jang, GlenManglapus, and Joyce Qi. Thanks alsoto my long-time collaborator and col-league, Steven Youngentob.

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Schwob 2002 the Anatomical Record