Cell Dynamics in the Embryonic and Postnatal VomeronasalEpithelium of SnakesDAVID A. HOLTZMAN*Department of Brain and Cognitive Sciences, University of Rochester, Rochester, New York 14627

KEY WORDS neurogenesis; cyclin-dependent kinases; cell cycle; development; olfactoryepithelium

ABSTRACT This review will discuss changes observed in the cell dynamics of the vomeronasalepithelium (VNE) of snakes during embryonic and postnatal growth. Recent work suggests thatneuronal differentiation occurs early in VNE development. We have used an antibody to anevolutionarily conserved peptide sequence (the PSTAIRE region) in a family of cell cycle regulatoryproteins, the cyclin-dependent kinases, to identify neuronal precursors in the embryonic andpostnatal VNE. During prenatal development, the location of neuronal precursors changes in theVNE. Significant postnatal changes occur in cell proliferation in the VNE (as determined by3H-thymidine autoradiography) and possibly in the larger complement of VNE receptor cellprecursors (as determined by anti-PSTAIRE staining). A model is proposed for changes in cellproliferation and death during embryonic development and postnatal maintenance and senescencein VNE of snakes, which may be applicable to the VNE and olfactory epithelium of other vertebrates.Microsc. Res. Tech. 41:471–482, 1998. r 1998 Wiley-Liss, Inc.

INTRODUCTIONThe vomeronasal system (VNS) of snakes is one of the

best-described chemosensory systems in vertebrates.The VNS of snakes is hypertrophied compared to theVNS of other vertebrates, and many studies haveshown the importance of this system in mediatingnaturally occurring behaviors in snakes (see Halpern,1992; Stone and Holtzman, 1996). The VNS of snakeshas been shown to be important for mediating court-ship and mating (as in mammals), aggregation, trailingof conspecifics and prey, prey attack, and ingestion. Inaddition, a wealth of data has accumulated on theanatomical, physiological, cellular, and molecular de-tails of the ophidian VNS (see Halpern 1992; Inouchi etal., 1993; Jiang and Terashima, 1996; Liu et al., 1997;Luo et al., 1994; Wang et al., 1993, 1997). Developmentof the VNS is of particular interest in snakes (Holtz-man, 1993). Newborn garter snakes, without experi-ence with food or food odors, show preferential re-sponses to aqueous extracts of natural prey (reviewedin Burghardt, 1970, 1993). These responses were latershown to be mediated, in part, by the VNS (Burghardtand Pruitt, 1975). In addition, neonatal snakes exhibitother behaviors, such as aggregation (Burghardt, 1983;Ten Eyck and Halpern, 1988) and preferences forconspecific chemicals (Graves and Halpern, 1988; Hellerand Halpern, 1982a,b), which have been shown to bemediated by the VNS in adult snakes. These results arethe only evidence of vomeronasal function in neonatalvertebrates.

This review examines the changes observed in theprenatal and postnatal VNE of snakes. Initial findingsfrom gross morphological studies are confirmed andextended with information from 3-µm-thick sections.Tritiated thymidine autoradiography has been used toexamine the origins of receptor and supporting cell

precursors and the survival of labeled cells in theembryonic and neonatal garter snake, in vivo and invitro (Holtzman and Halpern, 1991). Results from thesestudies with 3H-thymidine autoradiography are con-firmed and extended with a marker for a family of cellcycle regulatory proteins, the cyclin-dependent kinases.Together, these data suggest that distinct periods existfor the expansion of neuronal precursor cells, neuronaldifferentiation, and the loss of neuronal precursor cells.A predictive model is proposed for changes in cellproliferation and death during embryonic developmentand postnatal maintenance and senescence in the VNEof snakes.

EMBRYONIC DEVELOPMENTIt is difficult to estimate the time of conception in

snakes because females are capable of storing sperm forlong periods of time (Hoffman, 1968). Embryonic snakesare characterized by external characteristics (Zehrstages, described by Zehr, 1962) and morphologicalcharacteristics of the vomeronasal and olfactory sys-tems (Holtzman and Halpern, 1991). The gross morpho-logical changes and 3H-thymidine autoradiographicresults described below have been reviewed previouslyin detail (Holtzman, 1993). As in the adult (Wang andHalpern, 1980a,b), the VNE of neonates consists of tallcolumns of bipolar receptor cells covered at the lumenalsurface by a thin layer of supporting cells (Holtzmanand Halpern, 1990, 1991). Undifferentiated cells arelocated at the base of each column and give rise to newreceptor cells (Wang and Halpern, 1982a,b, 1988). As

Contract grant sponsor: NCRR; Contract grant number: 08700.*Correspondence to: David A. Holtzman, Department of Brain and Cognitive

Sciences, University of Rochester, 105 Meliora Hall, Rochester, NY 14627.E-mail: holtzman@bcs.rochester.edu

Received 26 March 1998 ; accepted in revised form 4 May 1998

MICROSCOPY RESEARCH AND TECHNIQUE 41:471–482 (1998)

r 1998 WILEY-LISS, INC.

described below, the highly organized structure of thepostnatal VNE develops primarily during embryogen-esis.

Early Gestation: Zehr Stages 23–30The VNE of snakes is derived from the olfactory

placode, as shown for other reptiles (reviewed in Holtz-man, 1993). At approximately one-quarter throughgestation (Zehr stage 23), the VNE begins as a medialinvagination from the olfactory placode and appears asa single thick cell layer. By one third through gestation(Zehr stage 28), small round clusters of cells are seendorsal and posterior to a thicker cell layer adjacent tothe lumen of the VNE (Fig. 1). As for all later ages (seebelow), blood vessels are found between the basal andlumenal cell layers (Figs. 1 and 2). As described below,the basal cell layer will ultimately grow into the tall cellcolumns of the postnatal VNE, and the lumenal celllayer will become the thin supporting cell layer. Three-micrometer-thick sections of the Zehr stage 23 VNEshow that the small clusters of cells contain roundnuclei and mitotic figures (Fig. 2). Rounded, dark-staining, and shrunken nuclei, characteristic of pykno-sis, appear primarily in the lumenal layer (Fig. 2). Theapical half of the lumenal layer is also characterized byelongated nuclei, presumptive supporting cells, andoccasional mitotic figures (Fig. 2).

Receptor cell precursors in the VNE can be stained byan antibody to the PSTAIRE region of cyclin-dependentkinases (Holtzman and Clarke, 1993; Holtzman et al.,1996). All known cyclin-dependent kinases share iden-tity in 16 amino acids (EGVPSTAIREISLLKE) calledthe PSTAIRE region (Elledge and Spottswood, 1991;Lewin, 1990; Pines, 1993). These kinases help regulateprogression through the cell cycle in all cell typesexamined by associating with other cell cycle regula-tory proteins, namely cyclins (Lewin, 1990; Pines,1993). The PSTAIRE antigen should be expressed in allcells capable of cell division and has been used to showchanges in the postnatal VNE of snakes (Holtzman andClarke, 1993; Holtzman et al., 1996; and see below). Inthe VNE of Zehr stage 28 embryos, anti-PSTAIREimmunoreactivity is present in most, but not all cells, ofthe small cell clusters (Fig. 3). PSTAIRE is also ex-pressed in some cells along the lumen (Fig. 3). Theseresults suggest that cells capable of cell division can befound in cells from both basal and lumenal regions.This coincides with 3H-thymidine studies that show cellproliferation in both layers and suggests that precur-sors for receptor and supporting cells are segregated(Holtzman and Halpern, 1991).

The presence of young neurons in the Zehr stage 30VNE has been confirmed using antibodies to the growth-associated protein GAP-43/B50 (generously providedby Dr. K. Meiri). Anti-GAP-43/B50 antibodies stain thecell bodies and axons of immature neurons of theolfactory epithelium (Meiri et al., 1991; Verhaagen etal., 1990). In the Zehr stage 28 VNE, cell bodies arestained in both the basal cell columns and lumenallayer (Fig. 4). Vomeronasal axons are also stainedheavily (Fig. 4). These results show that young neuronsare present early in the embryonic VNE and are notrestricted to the basal cell clusters. A comparison ofanti-PSTAIRE and anti-GAP-43 staining shows thatboth precursors and differentiating cells are present in

the basal cell clusters (compare Figs. 3 and 4). Asexpected, no expression of GAP-43/B50 is present in theapical half of the lumenal layer, supporting the ideathat there is a spatial segregation of neuronal precur-sors and precursors for supporting cells (Holtzman andHalpern, 1991).

Late Gestation: Zehr Stages 32–37 (Birth)By mid-gestation (Zehr stage 32), short columns of

cells surround the lumenal layer of cells (Fig. 5).Three-micrometer-thick sections of the Zehr stage 32VNE show mitotic figures in the basal cell columns andapical half of the lumenal layer and pyknotic nuclei inthe lumenal layer (Fig. 6). As for the Zehr stage 28VNE, the lumenal layer consists of cells with roundnuclei (basal half) and elongated nuclei (apical half,Fig. 6). Mitotic figures in the lumenal layer appearrestricted to the region directly adjacent to the lumen.Anti-PSTAIRE immunoreactivity is seen primarily atthe base of the receptor cell columns, and staining isalso present along the lumen (Fig. 7). Similar patternswere seen for cells that incorporate 3H-thymidine atthis age in the lumenal layer and cell columns (Holtz-man and Halpern, 1991). The spatial segregation ofcells labeled by 3H-thymidine and the anti-PSTAIREantibody supports the idea that supporting cell precur-sors are located along the lumenal surface while neuro-nal precursors become restricted to the base of the basalcell columns (Holtzman and Halpern, 1991; and seebelow).

The gradual decrease of the lumenal layer corre-sponds with a gradual increase in the receptor cellcolumns in Zehr stage 32–37 embryos. Based on previ-ous gross morphological studies in snakes (Holtzmanand Halpern, 1990; Parsons, 1970), the increase in cellcolumn size may be due to either cell proliferationand/or cell migration from the cells in the lumenal layerto the growing columns. Cell turnover in the embryonicsnake VNE was demonstrated by long-survival studiesusing 3H-thymidine (Holtzman and Halpern, 1991),and the data from 3-µm-thick sections suggest that celldeath is restricted mostly to the lumenal layer duringlate VNE embryogenesis (Figs. 2 and 6). In the embry-onic (Holtzman and Halpern, 1991), neonatal (Holtz-man and Clarke, 1993), and adult (Wang and Halpern,1982a,b, 1988) VNE of snakes, neurons move apicallyas they age. Neurons also move apically during VNEturnover in adult rodents (Barber and Raisman 1978a,b;Wilson and Raisman, 1980). The presence of pyknoticnuclei near the border of the basal and apical halves ofthe lumenal layer may represent cells of a neuronallineage that are turning over. Alternatively, these cellsmay be from a non-neuronal lineage that are pro-grammed to die. Ultrastructural studies on the embry-onic VNE of snakes are needed to identify these cells.

Data from 3H-thymidine autoradiographic experi-ments show that cell proliferation occurs in the smallclusters of the Zehr stage 28 VNE but becomes re-stricted to the base of each column after Zehr stage 32(Holtzman and Halpern, 1991). Thus, neurogenesis inthe VNE appears to become restricted to the base of thereceptor cell columns with age (see Fig. 14 in Holtzmanand Halpern, 1991). During the first half of gestation(up to Zehr stage 32), neuronal precursors are foundthroughout the basal cell layer. As the basal cell layer

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Fig. 1. Vomeronasal epithelium (VNE) of a Zehr stage 28 snakeembryo. Notice small, round clusters of cells at the base of theepithelium (basal cell clusters, BL) and the thick cell layer (lumenallayer, LL) adjacent to the lumen (L). Arrowhead denotes pyknoticnucleus in the lumenal cell layer. Unless otherwise specified, allfigures of VNE sections were cut at 10 µm in the horizontal plane, withrostral at the top and medial to the right. Cresyl violet stain. bv, bloodvessel. Bar 5 12 µm for Figures 1–3.

Fig. 2. Zehr stage 28 VNE cut at 3 µm. Large arrowheads denotemitotic figures in the small, round clusters at the base and along thelumen. Notice that the basal half of the lumenal layer consists mostlyof cells with rounded nuclei while the apical half contains cells with

elongated nuclei. Small arrowheads denote pyknotic nuclei along thelumen. Toluidine blue stain. bv, blood vessel.

Fig. 3. Zehr stage 28 VNE stained with an antibody for thePSTAIRE antigen conserved among cyclin-dependent kinases, cellcycle regulators. Notice heaviest immunoreactivity (i.e., darkenedregions of VNE) in many, but not all, cells in the small, round clustersat the base and along the lumen. Preabsorption of the antiserum withp34cdc2 (a cyclin-dependent kinase) results in an absence of immunore-activity (data not shown), suggesting staining specificity for cyclin-dependent kinases. The anti-PSTAIRE immunoreactivity correspondsto regions containing cells capable of dividing.

473VOMERONASAL EPITHELIUM DEVELOPMENT IN SNAKES

grows from the round clusters to tall columns, neuronalprecursors are thought to become restricted to the mostbasal portion of the columns (as supported by PSTAIREexpression, Fig. 7) while differentiating daughter cellsoccur more apically. During development, precursorcells at the base of the VNE appear to generate twosubsets of daughter cells. One set, the ‘‘early’’ migratingsubpopulation, moves apically to form the columnspresent by Zehr stage 32. The other set of daughtercells, the ‘‘late’’ migrating subpopulation, remain at thebase of the VNE columns and move apically during thelater stages of embryogenesis. Subpopulations of ‘‘early’’and ‘‘late’’ differentiating neurons have also been de-scribed in the developing olfactory epithelium (Calofand Chikaraishi, 1989; Cushieri and Bannister, 1975a,b;Gordon et al., 1995; Mackay-Sim and Kittel, 1991;Paternostro and Meisami, 1993; Suzuki and Takeda,1993) and developing cortex (Takahasi et al., 1996a,b)of rodents. By birth, the VNE is very similar to theadult VNE where neuronal precursors are restricted tothe basal layer of the columns and give rise to onlyreceptor cells (Holtzman and Clarke, 1993; Holtzman etal., 1996; Wang and Halpern, 1980a,b, 1982a,b, 1988).This model is also supported by the restriction ofPSTAIRE expression to the basal regions of the recep-tor cell columns from Zehr stage 30 (Fig. 7) to neonates(Holtzman and Clarke, 1993; Holtzman et al., 1996)and adults (see Fig. 9).

POSTNATAL CHANGESTo investigate changes in VNE cell dynamics during

normal aging, the overall population of precursor cellshas been distinguished from the subset of activelydividing precursors cells. Cell dynamics were examinedwith the following assumptions: (1) 3H-thymidine auto-radiography with short survival times will identify cells

in the DNA-synthesis phase of the cell cycle at the timeof injection; (2) Immunocytochemistry with an antibodyto the PSTAIRE region of cyclin-dependent kinasesshould identify all neuronal precursors capable of divid-ing; and (3) Combined 3H-thymidine autoradiographyand anti-PSTAIRE immunocytochemistry will estimatethe percentage of neuronal precursors that are dividingin the tissue. The data from these experiments gives usa ratio of basal cells to dividing precursors for the VNEof juvenile and adult garter snakes (Holtzman andClarke, 1993; Holtzman et al., 1996).

Cell Proliferation in Neuronal PrecursorsObservations after survival times of 1 hour, 1 day, 1

week, 1 month, and 2 months post-3H-thymidine injec-tion for 2–3-month-old snakes, show that cells labeledby 3H-thymidine migrate from the basal layer of theVNE (1 hour and 1 day survivals) into the receptor cellcolumns (1 week–2 months survivals, Holtzman andClarke, 1993; Holtzman et al., 1996). Two months after3H-thymidine injection, some labeled cells are seen inthe basal layer of the VNE with most in the receptor celllayer. This finding is similar to the presence of ‘‘early’’and ‘‘late’’ differentiating subpopulations of daughtercells from neuronal precursors identified in the VNE ofembryonic garter snakes (Holtzman and Halpern, 1991;and see above). There is a significant statistical in-crease in receptor cell number in the garter snake VNEfrom 2–3 months of age (126.40 6 7.15) to adulthood(186.60 6 5.44, Mann-Whitney U-test, P , 0.05, Holtz-man et al., unpublished results; and compare Figs. 5and 8). However, 1 hour after 3H-thymidine injection,the adult VNE has approximately 70% fewer cellslabeled by 3H-thymidine in the basal layer than seen inthe basal layer of 2–3-month-old snakes (Holtzman etal., 1996).

Fig. 4. Zehr stage 30 VNE stained with an antibody for GAP-43/B50, a growth-associated proteinfound in immature neurons. Notice the heavy staining in the vomeronasal nerve fascicles (arrowheads)and cells in the small, round clusters at the base of the VNE. Cell bodies are also stained in the basal halfof the lumenal cell layer. Bar 5 12 µm.

474 D.A. HOLTZMAN

Cyclin-Dependent Kinase Expressionin Neuronal Precursors

Anti-PSTAIRE immunoreactivity was expected toidentify all cells within all phases of the cell cycle.Anti-PSTAIRE immunoreactivity is seen only at thebase of the VNE for all postnatal animals (Fig. 9,Holtzman and Clarke, 1993; Holtzman et al., 1996).Three to four times more precursor cells in youngsnakes are labeled consistently by the PSTAIRE anti-body than by 3H-thymidine (Holtzman and Clarke,1993; Holtzman et al., 1996). The number of anti-PSTAIRE immunoreactive cells is significantly greaterin 2–3-month-old snakes than in snakes 3–4 months oldthrough adulthood (Holtzman and Clarke, 1993; Holtz-man et al., 1996). As for cells labeled by 3H-thymidine,adult snakes have approximately 70% fewer cells ex-pressing PSTAIRE than 2–3-month-old snakes (Holtz-man et al., 1996).

Anti-PSTAIRE immunoreactivity was examined inrelation to 3H-thymidine labeling 1 hour post-3H-thymidine injection (Holtzman and Clarke, 1993; Holtz-man et al., 1996). In sections from young and adult

snakes double-labeled by 3H-thymidine and anti-PSTAIRE, all double-labeled cells are found in thebasal layer of the VNE (Fig. 10). Of all cells labeled by3H-thymidine, 85.7% are anti-PSTAIRE immunoreac-tive at 1 hour post-3H-thymidine injection in 2–3-month-old snakes, suggesting that only a subset of the totalprecursor cell population is anti-PSTAIRE immunoreac-tive (Holtzman et al., 1996). Nearly 100% of all cellslabeled by 3H-thymidine are anti-PSTAIRE immunore-active in adult snakes (Holtzman et al., 1996; Fig. 10).However, 23% of all anti-PSTAIRE immunoreactivecells are also labeled by 3H-thymidine 1 hour post-injection in both the 2–3-month-old and adult snakes(Holtzman et al., 1996), suggesting a maintenance ofactive cell division relative to the total population ofprecursor cells. From 2 months of age to adulthood,there is approximately a 70% decrease in both thenumber of precursors that are actively dividing duringnormal turnover and in the total number of precursorcells in the snake VNE (Holtzman and Clarke, 1993;Holtzman et al., 1996).

Fig. 5. VNE of a Zehr stage 32 embryo. Notice the formation ofcolumns of cells which are approximately equal in thickness to thelumenal cell layer. Cresyl violet stain. bv, blood vessel. Bar 5 12 µm forFigures 5 and 6.

Fig. 6. Zehr stage 32 VNE cut at 3 µm. Large arrowheads denotemitotic figures in the small, round clusters at the base and along the

lumen. Notice that the cell columns and basal half of the lumenal layerconsist mostly of cells with rounded nuclei while the apical half of thelumenal layer contains cells with elongated nuclei. Small arrowheadsdenote pyknotic nuclei in the middle of the lumenal cell layer, near theborder of cells with round and elongated nuclei. Toluidine blue stain.bv, blood vessel.

475VOMERONASAL EPITHELIUM DEVELOPMENT IN SNAKES

If there is maintenance of the number of dividingcells relative to the total complement of precursor cellsin the postnatal VNE, we can hypothesize the changesthat would need to occur in receptor cell numbersduring postnatal life. Figure 11 shows a model summa-rizing the changes that would be expected with 25% ofprecursor cells giving rise to neurons from 2–3 monthsto adulthood in snakes. Although a few precursors inthe VNE give rise to daughter cells that remain in thebasal cell layer of the receptor cell columns while themajority move apically (as mentioned above for theVNE of embryos and 2–3-month-old snakes), it is notknown if the daughter cells that remain at the base ofthe columns continue to divide or differentiate laterwithout undergoing more rounds of cell division. There-fore, we can assume that the minimal number ofneurons that could be produced by a precursor cell(minimum neuronal potential) will be two neurons. Asshown in Figure 11, the model predicts that there wouldbe a 50% reduction in the minimum number of neuronsproduced as the number of precursors is reduced byhalf. Assuming a 75% reduction from birth to adult-hood, the model predicts that the minimum neuronalpotential of adults will be one quarter of the minimumneuronal potential of newborns. Recently, we havedetermined that the number of receptor cells increasesby approximately 50% in the VNE of snakes from 2–3months to adults (Holtzman et al., 1996). Taken to-gether, the predictions from the model and data onreceptor cell numbers suggest that there is increasedreceptor cell survival with age. A corollary of thishypothesis is that normal turnover of receptor cells inthe snake VNE may slow down with age.

CHANGES WITH AGE IN THE CELLDYNAMICS OF THE VNE:A PREDICTIVE MODEL

Given that the VNE arises from the olfactory placode(Brunjes and Frazier, 1986; Holtzman and Halpern,1990), it is probable that the VNE and olfactory epithe-lium share mechanisms that regulate cell proliferationand turnover. Evidence for proliferating and differenti-ating subpopulations of neuronal precursors comesfrom studies of the rodent olfactory epithelium (Calofand Chikaraishi, 1989; Cushieri and Bannister, 1975a,b;Gordon et al., 1995; Mackay-Sim and Kittel, 1991;Paternostro and Meisami, 1993; Suzuki and Takeda,1993) and the garter snake VNE and olfactory epithe-lium (Holtzman and Halpern, 1991). In the olfactoryepithelium of rodents, a specific subpopulation of basalcells, the globose basal cells, have been shown to giverise to neurons during development (Suzuki and Tak-eda, 1993), normal turnover in adults, and in responseto disruption (Breipohl et al., 1986; Caggiano et al.,1994; Goldstein and Schwob, 1996; Huard and Schwob,1995; Schwartz-Levy et al., 1991). Following olfactoryor vomeronasal system disruption, there is an increasein basal cell division in the olfactory epithelium(Breipohl et al., 1986; Caggiano et al., 1994; Carr andFarbman, 1992; Graziadei and Metcalf, 1971; Grazia-dei and Monti-Graziadei, 1979; Huard and Schwob,1995; Moulton et al., 1970; Schwartz-Levy et al., 1991;Schwob et al., 1992, 1995) and VNE (Wang and Hal-pern, 1982a,b, 1988), respectively. Decreases in neuro-genesis have been shown to result in the olfactoryepithelium from nasal occlusion in rodents (Farbman etal., 1988) and with increasing age (Loo et al., 1996;Paternostro and Meisami, 1994; Walker et al., 1990).

Fig. 7. Zehr stage 32 VNE stained with an antibody for thePSTAIRE antigen. Notice heaviest immunoreactivity mostly along thebase of the cell columns and in the apical half of the lumenal cell layer.This pattern of PSTAIRE expression suggests that the anti-GAP-43/

B50 immunoreactive cells with round nuclei in the basal half of thelumenal cell layer are not capable of cell division (see Figs. 4 and 6).Bar 5 12 µm.

476 D.A. HOLTZMAN

The data from garter snakes provides evidence for thepreservation of a specific percentage of neuronal precur-sor proliferation (approximately 25%) with age-relateddecreases in precursors within the cell cycle.

Our studies (Holtzman and Clarke, 1993; Holtzmanet al., 1996; unpublished results) suggest that there isdevelopmental regulation of cyclin-dependent kinaseexpression in the VNE of snakes which may be related

to changes in proliferation, differentiation, and survivalof neuronal precursors and their progeny (Fig. 12).Data support the occurrence of a proliferation phaseduring early VNE development (Holtzman and Hal-pern, 1991). The number of precursor cells increasesduring this phase with little or no production of neu-rons (Fig. 12). These precursor cells would correspondto a proliferating subpopulation. During early differen-

Fig. 8. The VNE of an adult garter snake. Notice the tall cellcolumns of receptor cells that are still separated from the apicalsupporting cell layer by blood vessels (bv). Pigment is abundant at thebase of the VNE and between cell columns. The extent of VNEpigmentation appears to increase gradually after birth (Holtzman,unpublished observations), and its significance is not known. Cresylviolet stain. Bar 5 24 µm for Figs. 8 and 9 and 12 µm for Fig. 10.

Fig. 9. Adult snake VNE stained with an antibody for the PSTAIREantigen. Notice that staining is restricted to a subset of cells at the

very base of the cell columns (arrowheads). In normal adults, anti-PSTAIRE immunoreactivity is only found in this location.

Fig. 10. Double labeling of a basal cell by the anti-PSTAIREantibody and 3H-thymidine injected 1 hour prior to sacrifice (largearrowhead). Several other cells labeled only by the anti-PSTAIREantibody can also be seen (small arrowheads). In the adult VNE,almost all cells labeled by 3H-thymidine injected 1 hour prior tosacrifice also express PSTAIRE.

477VOMERONASAL EPITHELIUM DEVELOPMENT IN SNAKES

tiation (Fig. 12), precursor cell numbers would bemaintained while neuronal populations increase. Earlydifferentiation has occurred certainly by Zehr stage 30,as evidenced by the presence of anti-GAP-43/B50 immu-noreactive cells (Fig. 4). Daughter cells of precursors,which differentiate into neurons, would correspond to adifferentiating subpopulation. Maintenance of precur-sor cell number could result from: (1) a population ofprecursors that give rise to daughter cells, one of whichremains undifferentiated with the other differentiat-ing; and/or (2) a population of mixed precursors thatgive rise to only other precursors or only cells thatdifferentiate. Both of these possibilities are supportedby the presence of cells labeled by 3H-thymidine in thebasal layer of young snakes two months after injectionof the tracer (Holtzman and Clarke, 1993; Holtzman etal., 1996).

During a late differentiation phase (Fig. 12), therewould be a net decrease in the number of precursor cellsand maintenance of neuronal number. Two scenarioscould account for this. In one case, there would be apopulation of mixed precursor cells. Some cells wouldgive rise to an additional precursor cell and a cell thatdifferentiates, and other cells would give rise to onlycells that differentiate. In this scenario, differentiating

cells would have to die to yield a net maintenance ofneuronal number. The second scenario involves a singlepopulation of precursor cells that give rise to bothprecursor and differentiating cells. In this case, one ofthe precursor cells generated would have to die in orderfor there to be a net decline in this population. Thedeath of differentiated neurons is suggested by thepresence and location of pyknotic nuclei in the VNE inthe Zehr stage 32 embryo (Fig. 6). Current data fromthe developing olfactory epithelium support both sce-narios. Immature neurons are known to die duringnormal growth (Hinds et al., 1984; Holcomb et al.,1995), and cell death has been reported in the neuronalprecursor cell layer of the olfactory epithelium (Hol-comb et al., 1995). In the senescence phase (Fig. 12),there would be elimination of bipotent precursor cells,with net decreases of neurons. This could be achieved intwo ways: (1) unipotent precursor cells that only giverise to differentiating cells, or (2) bipotent precursorcells that give rise to a unipotent precursor cell and adifferentiating cell. Both possibilities are supported bydata from long survival 3H-thymidine studies. Cellsthat incorporate 3H-thymidine have been found in thebasal layer of the VNE in adult snakes, as late as 63days after injection (Halpern, unpublished results).

Fig. 11. Model for age-related changes inthe numbers of neuronal precursors and theminimum number of neurons they can pro-duce (see text for details). Data from com-bined anti-PSTAIRE immunocytochemistryand 3H-thymidine autoradiography suggeststhat approximately one quarter of neuronalprecursors are normally dividing in the post-natal VNE of snakes. As snakes age from afew months to adulthood, the number ofprecursor cells decreases, but the ratio ofdividing cells (one quarter) remains the same.According to this model, fewer neurons will beproduced with age.

478 D.A. HOLTZMAN

The models presented in Figures 11 and 12 accountfor the postnatal decrease of precursor cells, describedhere for the VNE of snakes and noted previously in theolfactory epithelium of rodents (Loo et al., 1996; Pater-nostro and Meisami, 1994; Walker et al., 1990). Themodel can now be tested by quantifying the generationand cell death of precursor cells and neurons. Wepredict that there are changes in cell cycle dynamics ofthe precursor cell population, during transitions fromearly embryonic growth to postnatal growth, mainte-nance, and aging. As shown in the current observations,valuable information can be obtained about cell dynam-ics by looking at relationships between subpopulationsof precursors. We are currently investigating the rela-tionships between these subpopulations in response tovomeronasal nerve lesion. Changes in the ratio be-tween actively dividing cells and anti-PSTAIRE immu-noreactive cells may point to specific regulation of asubpopulation of precursor cells. Thus, the model wehave presented should be useful for explaining changes

in cell proliferation and differentiation within the VNEafter vomeronasal system disruption. A natural experi-ment of vomeronasal system disruption can be seen ingarter snakes with abnormal VNEs (Figs. 13–15). Thecause of this disruption is not known, but it may beinherited or transmitted from mothers to their embryosbecause this disorder has been seen among littermates,but not conspecifics, born and raised concurrently(Holtzman et al., unpublished results). The cells in thebasal half of the receptor cell columns appear largerthan normal and are not stained well by Nissl stains(Fig. 11). Interestingly, there is a striking increase inanti-PSTAIRE immunoreactivity in this abnormal VNE(Figs. 14 and 15). There is an increase in PSTAIREexpression along the base of the receptor cell columns,but there is also PSTAIRE expression in the apical halfof the receptor cell columns and in the supporting celllayer (Figs. 14 and 15). In normal animals, PSTAIREexpression is only seen in basal cells (see Fig. 9;Holtzman and Clarke, 1993, Holtzman et al., 1996).

Fig. 12. Model for the snake VNE, show-ing the transitions from its genesis to themaintenance of the receptor cells and aging invivo. A: In the Proliferation phase, a netexpansion of precursor cells is hypothesizedduring embryogenesis. B: During the EarlyDifferentiation phase, a net maintenance ofprecursor cell number is predicted with netexpansion of receptor cells (neurons). C: Dur-ing the Late Differentiation phase, a netdecrease in the precursor cell number is pre-dicted with a net maintenance of neuronalnumbers. D: In the Senescence phase, therewould be a gradual elimination of precursorcells with a net decrease in the number of newneurons generated. Cell death (X) is predictedfor daughter cells that either remain as pre-cursors or differentiate into neurons. Al-though represented as a single cell, the sym-bols represent populations of cells. See textfor further details.

479VOMERONASAL EPITHELIUM DEVELOPMENT IN SNAKES

These results suggest that disruption of the VNE mayresult in an upregulation in the number of precursorcells throughout the VNE.

Changes in the strategies used to generate precursoror differentiating cells during development or regenera-tion would affect the numbers of each cell type gener-

Fig. 13. Abnormal VNE of an adult garter snake. Cells at the baseof the cell columns appear swollen. Notice that these swollen cells donot stain well with cresyl violet. Bar 5 24 µm for Figs. 13 and 14 and12 µm for Fig. 15.

Fig. 14. VNE of the same snake from Figure 13 stained by theanti-PSTAIRE antibody. Notice the larger complement of immunoreac-tive cells at the base of the cell columns as compared to normal snakes

(see Figs. 9 and 10). In addition, cells expressing PSTAIRE are foundthroughout the cell columns and in the supporting cell layer along thelumen.

Fig. 15. Higher magnification of the abnormal VNE stained by theanti-PSTAIRE antibody. Virtually all cells along base of the cellcolumns are expressing PSTAIRE. Cells expressing PSTAIRE are alsoseen more apically in the cell columns (arrowheads).

480 D.A. HOLTZMAN

ated. These changes should help to identify whichspecific cell groups are subject to environmental influ-ence. We will be able to examine the role of specific geneexpression on the regulation of neurogenesis as moremarkers become available to better define the popula-tion of precursors and their progeny in reptiles.

ACKNOWLEDGMENTSI thank Dr. Mimi Halpern for her support and

encouragement over my entire career, and the manyundergraduates who have worked in my laboratoryover the years and for whose help I am forever grateful.I also thank Dr. Tim Smith and an anonymous reviewerfor helpful suggestions and comments and especiallythank Dr. Elizabeth Bostock and our family for provid-ing the best support imaginable. The research from mylaboratory has been supported by NCRR grant 08700.

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