Expression of rat target of the antiproliferative antibody (TAPA) in the developing brain

Expression of Rat Targetof the Antiproliferative Antibody(TAPA) in the Developing Brain

C.D. SULLIVAN AND E.E. GEISERT, JR.*

Department of Anatomy and Neurobiology, University of Tennessee,Memphis College of Medicine, Memphis, Tennessee 38163

ABSTRACTThe present study defines the expression pattern of TAPA (target of the antiproliferative

antibody, also known as CD81) in the developing rat brain. TAPA is a member of thetetramembrane spanning family of proteins, and like other members of this family it appearsto be associated with the stabilization of cellular contacts (Geisert et al. [1996] J. Neurosci.16:5478–5487). On immunoblots of the brain, TAPA is present in higher levels than any othertissue examined: muscle, tendon, peripheral nerve, cartilage, liver, kidney, skin, and testicle.Immunohistochemical methods were used to define the distribution of TAPA in the brain. Thisprotein is expressed by ependyma, choroid plexus, astrocytes, and oligodendrocytes. TAPA isdramatically upregulated during early postnatal development, at the time of glial birth andmaturation. At embryonic day 18, the levels of TAPA are low, with most of the immunoreactionproduct being associated with the ependyma, choroid plexus, and the glia limitans. Asdevelopment continues, the amount of TAPA expressed in the brain increases, and atpostnatal day 14 the levels approach those of the adult. This increase in the levels of TAPA atpostnatal day 14 is due to upregulation in the gray matter and white matter. Thus, TAPA isfound in all glial cells, and the level of this protein correlates with their maturation. J. Comp.Neurol. 396:366–380, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: CD81; astrocytes; oligodendroglia; central nervous system; cell adhesion

A major event in the final stages of central nervoussystem (CNS) development is the birth and maturation ofglia. The glial cells born during this period form themajority of cells in the CNS and have a number of differentfunctions. Within the brain and spinal cord, both microglia(Flaris et al., 1993) and macroglia are present. The presentreport is focused on the different types macroglia: astro-cytes, oligodendrocytes, ependymal cells, choroidal cells,and radial glia. The earliest generated glial cells are theradial glia (Misson et al., 1991). These cells are specificallyassociated with neuronal migration (Rakic, 1971; Cavinessand Rakic, 1978; Hatten, 1990; Misson et al., 1991; Ko-muro and Rakic, 1993). Shortly after the emergence of theradial glia, the ependymal lining of the ventricular systemdevelops and the choroid plexus forms (for review, see DelBigio, 1995). As neurogenesis is ending, the final popula-tions of glial cells are born during the late embryonic andthe early postnatal period (Ling and Leblond, 1973; Parna-velas et al., 1983). The majority of these cells differentiateinto astrocytes and oligodendrocytes.

As the astrocytes mature, they induce the formation of anumber of functional barriers such as the blood–brainbarrier. Astrocytes are also associated with the glia limi-

tans and the ependymal lining of the ventricular system.In addition, these cells regulate the levels of ions and otheractive molecules in the extracellular space, thus providinga homeostatic environment (Walz and Hertz, 1982; Balla-nyi et al., 1987). The maturation of astrocytes is reflectedin the regulated expression of specific proteins. Bothprotoplasmic astrocytes (found in the gray matter) andfibrous astrocytes (found in the white matter) expressvimentin early in development and glial fibrillary acidicprotein (GFAP) as they mature (Eng et al., 1971; Bignamiand Dahl, 1973; Dahl, 1981; Pixley and de Vellis, 1984;Weir et al., 1984). The transition from an immature to amature state is also marked by the expression of specificglycoconjugates that may be involved in pattern formationand the stabilization of neuronal contacts (Steindler et al.,

Grant sponsor: University of Tennessee Center for Excellence in Neurosci-ence; Grant sponsor: Spinal Cord Society.

*Correspondence to: Eldon E. Geisert, Jr., Ph.D., Department of Anatomyand Neurobiology, University of Tennessee at Memphis, 855 MonroeAvenue, Memphis, TN 38163. E-mail: [email protected]

Received 24 September 1997; Revised 6 March 1998; Accepted 11 March1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 396:366–380 (1998)

r 1998 WILEY-LISS, INC.

1990; Geisert and Bidanset, 1993). While the astrocytesare forming a stable CNS environment, they acquire thepotential to become reactive and form a glial scar (Berry etal., 1983). Many investigators believe that these reactiveastrocytes contribute to the lack of axonal regeneration inthe mammalian CNS (Reier and Houle, 1988).

Oligodendrocytes are the last major cell type born in thebrain. As these cells mature, they express many stage-specific antigenic markers. Oligodendrocyte progenitorsexpress the ganglioside GD3 (Goldman et al., 1984; Levi etal., 1987; LeVine and Goldman, 1988; Hardy and Rey-nolds, 1991). As maturation proceeds, a transitional periodis entered when GD3 and galactocerebroside (GC) arecoexpressed. As the cells continue to develop, GD3 expres-sion is lost and the expression of proteins marking matureoligodendrocytes begins (Hardy and Reynolds, 1991; Miller,1996). Oligodendrocytes are fully mature when they beginto myelinate axons. The compact layers of membranes thatform the myelin sheaths contain a number of uniquecomponents. Some components associated with myelin areGC (Raff et al., 1978, 1979), nucleotide-3-phosphohydrol-ase (McMorris, 1983), myelin basic protein (Reynolds andWilkin, 1988), myelin-associated glycoproteins (Salzer etal., 1990; Quarles et al., 1992), and myelin/oligodendrocyteglycoprotein (Linington et al., 1984). Mature oligodendro-cytes are essential for proper CNS function and arebelieved to prohibit axonal sprouting and growth (Caroniand Schwab, 1988).

We have recently identified a glial protein, the rat targetof the antiproliferative antibody (TAPA), that is a memberof the tetramembrane spanning family. As with othermembers of this family, TAPA appears to be associatedwith the formation of stable cellular contacts (Geisert etal., 1996b). The focus of the present study is to define thedevelopmental regulation of TAPA and its role in stabiliz-ing glial contacts.

MATERIALS AND METHODS

Immunohistochemistry

Postnatal Sprague-Dawley rats were anesthetized witha mixture of xylazine (13 mg/kg, Rompunt) and ketamine(87 mg/kg, Ketalart) that was administered by intraperito-neal injection. The animals were perfused through theheart with a solution of 0.1 M phosphate buffered saline(PBS; pH 7.5) followed by 4% paraformaldehyde in 0.1 Mphosphate buffer (PB; pH 7.5). All of the protocols used inthis study were approved by the Animal Care and UseCommittee of the University of Tennessee, Memphis. Thebrains and spinal cords were removed and placed in 4%paraformaldehyde for 4 hours. For cryoprotection, thetissues were placed in a 30% sucrose solution and allowedto sink. The brains from older rats were sectioned at 50 µmwith a freezing microtome (American Optical Corporation,New York, NY). The brains from younger animals were cutat 50 µm with a Vibratome (Technical Products Interna-tional, St. Louis, MO) or at 10 µm with a cryostat (ReichertHistostat, Buffalo, NY). Spinal cords were sectioned with afreezing microtome (American Optical Corporation). Allsections were stored in borate buffered saline (pH 8.4) at4°C.

To begin the immunohistochemical staining process, thesections were blocked for 2 hours at room temperaturewith 4% bovine serum albumin (BSA; Sigma, St. Louis,MO) in 0.2 M borate buffered saline (BBS; pH 8.4). The

sections were then placed in a solution containing theprimary antibody at a concentration of approximately20 µg/ml in 0.2 M BBS with 1% BSA. The sections wereincubated in this solution overnight at 4°C. Primaryantibodies included the AMP1 monoclonal antibody (di-rected against TAPA; Geisert et al., 1996b), the TuJ1monoclonal antibody (which stains class III b-tublin foundonly in neurons; Geisert and Frankfurter, 1989), and apolyclonal antibody directed against GFAP (Lipshaw, Pitts-burgh, PA). Sections were washed three times for 10minutes in BBS and then transferred to a solution contain-ing the secondary antibody for 2 hours at room tempera-ture. For mouse monoclonal antibodies, a peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) was used at adilution of 1:400 in BBS. For sections stained with rabbitpolyclonal antiserum, a peroxidase-conjugated donkey anti-rabbit IgG (Jackson Immuno Research Laboratories Inc.)was used at a dilution of 1:400. The sections were rinsed intwo changes of 0.2 M BBS, followed by three rinses in 0.1M PB (pH 7.2). Then the specimens were incubated in asolution containing 1 mg/ml of 3,3’-diaminobenzidine tetra-hydrochloride (Sigma) in 0.02 M PB (pH 7.4) and 5 µl/ml of3% hydrogen peroxide for 15–30 minutes at room tempera-ture. Controls included sections treated in a similar man-ner, with either the omission of the primary antibody orthe substitution of a nonimmune mouse IgG1 (ICN, CostaMesa, CA).

Gel electrophoresis and immunoblot method

Adult Sprague-Dawley rats were anesthetized with amixture of xylazine (13 mg/kg, Rompunt) and ketamine(87 mg/kg, Ketalart) and then decapitated. The brainswere removed from their skulls and homogenized in 5 ml of0.01 M PBS. Samples of different tissues were dissolved innonreducing sample buffer (2% sodium dodecylsulfate[SDS], 10% glycerol in 0.05 M Tris-HCL buffer, pH 6.8).Protein samples were taken from brains at embryonic day18 (E18), postnatal day 1 (P1), P7, P1, P30, and adult rats(300 g). Samples were separated by SDS–polyacrylamidegel electrophoresis with a 4–16% gradient mini-gel (Gei-sert et al., 1991) and transferred to nitrocellulose. Theblots were then blocked in 5% nonfat dry milk, probed withthe AMP1 antibody, anti-GFAP antiserum (Lipshaw), oranti-myelin basic protein antiserum (Boheringer Mann-heim, Indianapolis, IN). Then the sections were rinsed inborate buffer (pH 8.5), incubated in horseradish peroxida-se–labeled secondary antibody, and treated with diamino-benzidine and hydrogen peroxide, as previously described(Geisert and Bidanset, 1993). The level of immunoreactionproduct was determined by scanning the blots and analyz-ing these scans with the NIH Image Software. To definethe relative levels of TAPA expression, quantification ofproteins on the immunoblots was conducted by using asimple limiting dilution approach. All the samples werebalanced to a set standard by using NIH Image Software.The samples were then diluted in twofold increments, runon a 4–16% gradient gel, and transferred to nitrocellulose.The immunoblots were stained for TAPA by using theAMP1 antibody. Blots were scanned into the imagingsoftware with a Macintosh computer and a high-resolutiondesk top scanner (Hewlett Pakard ScanJet II CX). NIHImage software was then used to compare the intensity ofthe TAPA immunolabeling to define the dilution factor thatproduced a similar level of TAPA immunoreactivity. All

DEVELOPMENTAL EXPRESSION OF TAPA 367

samples were standardized to the adult by matching theintensity of the samples run on commassie blue-stainedgels by using a high-resolution desk top scanner (HewlettPakard ScanJet II CX) and NIH Image software.

Cell cultures

Glial cells were cultured from the cerebral cortex ofSprague-Dawley rat pups ranging in age from P1 to P5 byusing a protocol similar to that described in Geisert andStewart (1991). The animals were anesthetized by coldand decapitated. Immediately after decapitation, the brainswere removed. The meninges were stripped from thesurface, and the cortices were placed in a Petri dishcontaining 10 ml of Hank’s Balanced Salt Solution (HBSS).The tissue was placed in 20 ml of 0.1% trypsin in HBSS for10 minutes.

To define the distribution of TAPA, the cells weremechanically dissociated and placed in 30 ml of mediumcontaining either 5% or 10% fetal calf serum in 75-cm2

flasks at a density of 5 3 103 cells/cm2. The flasks wereplaced in an incubator at 37°C with 5% CO2. The cells werestained by using immunohistochemical methods when thecultures were approximately 70% confluent. The AMP1antibody was combined with a fluorescein-labeled goatanti-mouse secondary (Jackson Immuno Research Labora-tories Inc.) to identify TAPA expression. The cells werecounterstained with either a murine monoclonal IgMdirected against an oligodendrocyte antigen MOSP (Dyeret al., 1991) or a rabbit antiserum directed against GFAP(Lipshaw) to identify astrocytes. To facilitate GFAP stain-ing, 1% Triton X-100 was added to the BBS solution.Cultures were washed three times for 10 minutes in BBSand then transferred to a solution containing the second-ary antibody for 2 hours at room temperature. The second-ary antibodies, a rhodamine-conjugated donkey anti-mouse IgM (Jackson Immuno Research Laboratories Inc.)or a rhodamine-conjugated goat anti-rabbit IgG (JacksonImmuno Research Laboratories Inc.), were used at adilution of 1:500 in BBS. The cultures were rinsed threetimes for 10 minutes in BBS. The cultures were examinedwith a Leitz Laborlux D microscope.

In situ hybridization

Three adult Sprague-Dawley rats were perfused asdescribed above, with the exception that all of the solu-tions were made by using water treated with diethylpyrocarbonate. The brains were removed, placed in 30%sucrose, and sectioned at 20 µm on a cryostat. The sectionswere mounted directly onto glass slides and stored at220°C.

The probes were made from a 500-base-pair insertplaced into the Invitrogen pCR 2.1 vector. The insertencodes for bases 225–725 of the TAPA sequence (accessionnumber U19894). The sense and antisense cRNA probeswere made by using the Riboprobe kit from Promega(Madison, WI). The antisense and sense probes weresynthesized from the linearized plasmid by using the T7and SP6 promoters, respectively. The incorporation ofdigoxigenin (DIG) conjugated UTP was approximately 1DIG labeled to 4 unlabeled UTP.

The sections were removed from the freezer and placedat room temperature for 30 minutes. The sections werethen submersed in chloroform for 2 minutes to extract thelipids, and the chloroform was allowed to evaporate. Thesections were placed in a humidified chamber at 55°C for

30 minutes. The probe was prepared by adding 100 µl ofthe hybridization solution (23 standard saline citrate[SSC], 10% dextran sulfate, 2 mM EDTA, 50% deionizedformamide, and 200 µg of sperm DNA) to 200 ng/ml ofDIG-labeled probe. The mixture was warmed to 95°C for 4minutes. Then the labeled probe was added to the sections,and the solution was sealed by applying a coverslip overthe sections. Care was taken not to trap air under thecoverslip. The sections were incubated at 70°C for 4–6hours. The coverslips were removed by soaking the slidesin 23 SSC overnight at room temperature. The sectionswere rinsed three times at 55°C with 50% deionizedformamide in 13 SSC, two times in 13 SSC at roomtemperature, and five times in Tris buffered saline (TBS;pH 7.5) at room temperature. The sections were incubatedin the Boerhinger Mannheim blocking solution for 30minutes at room temperature. An anti-DIG antibody conju-gated to alkaline phosphatase (Boerhinger Mannheim)was then added to the solution at a 1:400 dilution with 1%blocking solution in TBS, pH 7.5, and incubated overnightat 4°C. The sections were rinsed four times for 10 minuteseach with TBS, pH 7.5. The nitro blue tetrazolium chlorideand 5-bromo-4-chloro-3-indolylphosphate toluidine-salt so-lution was added to the sections and allowed to react for8–16 hours in the dark at room temperature.

RESULTS

In the present study the monoclonal antibody AMP1 wasused to define the levels and distribution of TAPA (CD81).When protein samples were denatured with SDS andpresented on nitrocellulose, the AMP1 antibody recognizedtwo proteins. Both proteins were cloned and sequenced(Geisert et al., 1996b). The 106-kDa antigen was alphaactinin and the 27-kDa antigen was TAPA. Although theAMP1 recognized these two proteins when they weredenatured and presented on nitrocellulose, it only recog-nized TAPA in formalin-fixed tissue or in cultured cells.This finding is supported by several different lines ofevidence (Geisert et al., 1996a). Both alpha actinin andTAPA are found in detergent extracts of rat brain andprimary cultures of rat glia; however, the AMP1 antibodywill only immunoprecipitate TAPA, not alpha actinin(Geisert et al., 1996b). Furthermore, the AMP1 antibodyrecognizes alpha actinin on immunoblots of protein fromtissues it will not stain. For example, on immunoblots ofC6 glioma, human glioma (U373), or human brain, theAMP1 antibody recognizes a single protein at 106 kDa (ourantibody does not recognize human TAPA-1). When theseglioma or sections of human brain are stained with immu-nohistochemical methods, no immunoreaction product isobserved (data not shown), demonstrating that the anti-body can see alpha actinin on immunoblots but not intissues. Taken together, these data demonstrate that theAMP1 antibody recognizes only TAPA in sections of the ratbrain or in cultures of rat cells.

The relative levels and distribution of TAPA in differenttissues were examined by using immunoblot methods.Protein samples were taken from a number of different rattissues: liver, muscle, tendon, kidney, testicle, cartilage,skin, sciatic nerve, and brain. These protein samples werebalanced and placed in nonreducing sample buffer. Thehighest levels of TAPA were found in the protein samplesof brain, with detectable levels observed in the proteinsamples of cartilage, testicle, and kidney (Fig. 1). The

368 C.D. SULLIVAN AND E.E. GEISERT, JR.

remaining tissues had minimal levels of TAPA, includingthe sample of peripheral nerve (Fig. 1, lane G). Therelative levels of TAPA in the brain and kidney werecompared by a limiting dilution analysis. Approximatelyequivalent levels of TAPA were found when the full-strength sample of kidney proteins was compared with thesample of the brain diluted 1:64. These data demonstratedthe high levels of TAPA in the brain, especially in compari-son with the other body tissues examined.

Developmental regulation of TAPAin the brain

The developmental expression pattern of TAPA in thedeveloping brain was quantified by using immunoblot meth-ods. To allow for the quantitative immunoblot analysis ofTAPA, protein samples from different developmental stageswere made (Fig. 1, lanes A, B). Protein samples were takenfrom E18 (the time of peak neuronal birth in the cerebralcortex), P1 (near the end of neurogenesis and the beginning ofgliogenesis), P7 (a time near the peak of gliogenesis), P14 (atime of rapid glial maturation), and P30 (when the brain isrelatively mature). All of the samples were balanced, and thelevels of TAPA within each sample were determined by run-ning samples of two different ages on the same immunoblot.The levels of TAPA observed in the adult samples were set at100%, and three independent samples were taken at eachtime point (Fig. 2). The levels of TAPA observed at E18 and P0were similar and represented approximately 7% of the adult.The most dramatic upregulation of TAPA occurred in thesecond postnatal week, increasing from 15% of adult at P7 to70% by P14. After P14, there was a gradual increase untiladult levels were reached.

Because the increase in the levels of TAPA appeared tocorrelate with the birth and maturation of glial cells, weexamined the levels of proteins associated with astrocyteand oligodendrocyte maturation. As astrocytes mature,there is a switch from the intermediate filament proteinvimentin to GFAP. Thus, these two proteins were used asmarkers for the maturation of astrocytes. Immunoblots ofprotein samples from the developing brain were probedwith antibodies directed against GFAP and vimentin (Fig.3). As expected, the levels of vimentin decreased as thelevels of GFAP increased. This increase in GFAP anddecrease in vimentin occurs with approximately the sametime course as the upregulation of TAPA. The second majorclass of macroglia is the oligodendrocyte. Myelin basicprotein was used as a marker for mature oligodendrocytesand myelin (Fig. 3). The upregulation of myelin basicprotein was delayed relative to that of TAPA. Myelin basicprotein was not observed until P14. A dramatic increase inthe levels of myelin basic protein occurred between P14and P30. These immunoblot data indicate that TAPA isupregulated along a time course that is similar to that ofother proteins associated with glial maturation.

Cellular distribution of TAPA

Our immunoblot analysis indicates that TAPA is ex-pressed during the maturation of glia within the brain andthat very low levels of the protein are found before thisdevelopmental period. Three different approaches weretaken to define the cells within the brain that express thisprotein. The first was to examine cultured cells by usingimmunohistochemical methods. The second was to stainsections of the developing brain. The third was to use insitu hybridization to define the cells containing the mes-

Fig. 1. Immunoblots of protein samples were taken from differentrat tissues and stained with the AMP1 antibody directed against thetarget of the antiproliferative antibody (TAPA). Under nonreducingconditions, the AMP1 antibody recognizes two proteins when they arepresented on nitrocellulose: alpha actinin at 106 kDa and TAPA at 27kDa. Six different rat protein samples are shown: embryonic day 18(E18) brain (A), adult brain (B), dissected subcortical white matterfrom the adult (C), gray matter from the adult cerebral cortex (D), asample of cultured astrocytes (E), kidney (F), and peripheral nerve(G). The total load of proteins in all lanes were balanced. Notice thatthe levels of TAPA in the adult brain are considerably higher than thatobserved in the kidney, the embryonic brain, and peripheral nerve. Inaddition, the levels of TAPA in the white matter are approximatelyequal to that found in the gray matter. Molecular weight markers areindicated to the left in kilodaltons. This figure is an untouched digitalreproduction using Adobe Photoshop 3.0 (Adobe, Mountain View, CA).

Fig. 2. The levels of target of the antiproliferative antibody (TAPA)at different developmental ages are shown. Limiting dilutions ofbalanced protein samples were used to define the relative amounts ofTAPA, expressed as a percentage of adult. Three independent sampleswere measured at each developmental stage, represented by a square,circle and diamond. Through postnatal day 7 (P7), the expressionlevels of TAPA remain low, under 15%. By P14 a dramatic increase inexpression is seen, with 70% of adult expression observed. The changein expression between P7 and P14 is shown to be statistically differentby using a Mann-Whitney U test (P , 0.05). Notice that the levels ofTAPA continue to increase even after P30.


sage for TAPA. Neurons and glia were cultured from thedeveloping rat CNS and stained by immunohistochemicalmethods by using the AMP1 antibody. The cell cultureswere counterstained to define astrocytes (by using anti-GFAP antiserum), oligodendrocytes (by using the CE1monoclonal antibody), or neurons (by using the TuJ1antibody). AMP1 stained astrocytes and oligodendrocytes(Fig. 4). The TAPA immunoreaction product was seenprimarily at points of cell–cell contact for both type 1astrocytes and type 2 astrocytes. Oligodendrocytes weremore intensely stained by AMP1 than by astrocytes. Theentire surface of the oligodendrocyes were brightly stained,with staining extending from the cell bodies to the tips ofall processes. In these cultures, we never saw above-background levels of AMP1 immunostaining on the TuJ1-positive neurons (data not shown). These data demon-strate that both cultured astrocytes and culturedoligodendrocytes express TAPA. Several lines of evidencedemonstrate that the AMP1 monoclonal antibody recog-nized an antigen on the external surface of cultured glialcells. When the antibody is applied to living astrocytes for15 minutes, the antibody labels the surface of the culturedcells. This type of labeling also occurs within minutes ofexposure, even at 4°C. The pattern of labeling observedwhen living astrocytes are exposed to the antibody issimilar to the pattern observed when the cells are fixedand treated with detergents. These data indicate that themonoclonal antibody AMP1 recognizes an epitope of TAPAon the external surface of cultured glial cells.

To examine the cellular distribution of TAPA in thebrain, sections from the P14 rat cerebellum were stainedfor TAPA, astrocytes, and neurons (Fig. 5). The cerebellumwas chosen because of the distinctive difference in theorientation of glial processes relative to neuronal pro-cesses. Furthermore, at P14 the membranes of the Berg-mann glial cells have fewer fingerlike projections than are

observed in adult tissues. When a section was doublestained for GFAP and TAPA, the pattern of TAPA (Fig. 5A)was similar to that observed with GFAP (Fig. 5B). Thepattern of neurons seen in an adjacent section (Fig. 5C) isvery different from that of TAPA. These data support thehypothesis that TAPA is expressed by glia (astrocytes) andnot by neurons (Purkinje cells).

In situ hybridization

A second approach to define the cells within the adultbrain that express TAPA is to use in situ hybridization.Figure 6 illustrates the pattern of labeling in the hippocam-pus after hybridization with the sense (Fig. 6A,C) and theantisense probes (Fig. 6B,D) directed against TAPAmRNA.When comparing the antisense labeling with the senselabeling, a clear pattern of cellular labeling is observed. Inthe sections probed with the antisense, many small cellbodies are labeled in the sections. In addition, the choroidplexus is heavily labeled along with the ependymal liningof the ventricles. A closer examination of the tissues showsthat the large pyramidal cells of the hippocampus are notlabeled (Fig. 6B,D). In these sections, no labeling of largeidentifiable neurons was observed. The labeling of smallcell bodies within the gray matter and white matter is consis-tent with the expression of TAPAby glia within the CNS.

To provide for a direct comparison to the distribution ofmRNA in the hippocampus, additional sections throughthe hippocampus were stained for TAPA (Fig. 7A,C) and forGFAP (Fig. 7B,D). In the section stained for TAPA, thechoroid plexus and ependymal cells are heavily labeled. Inthe hippocampus, the overall pattern of TAPA staining(Fig. 7A) is similar to that observed in the section stainedfor GFAP (Fig. 7B). For example, there is a concentrationof GFAP (astrocytes) beneath the granular cell layer of thedentate gyrus. This increase in immunoreactivity is alsoobserved in the section stained for TAPA. The pattern ofTAPA immunoreactivity is consistent with the cellularexpression of TAPA mRNA as defined by in situ hybridiza-tion (Fig. 6).

Distribution of TAPA in the developing brain

To define the distribution of TAPA during the develop-ment of the CNS, we examined sections of the brain byusing indirect immunohistochemical methods. This analy-sis included coronal sections from a variety of developmen-tal stages: E18, P0, P7, P14, P30, and adult. In the E18brain, low levels of the AMP1 immunoreaction productwere observed within the parenchyma of the brain, withvirtually no labeling of the neocortex or neostriatum (datanot shown). The most notable accumulation of immunore-action product was observed in the choroid plexus and theependymal cell lining of the ventricular system. There wasalso a pronounced labeling of the glia limitans. Thepattern of AMP1 labeling at P0 is similar to that observedat E18 (Fig. 8B). Because the blood–brain barrier was notformed at this age, it was important to compare thepattern and intensity of labeling observed in the AMP1-stained sections with that found in similar control sectionstreated in an identical manner with the exception that theprimary antibody was omitted from the staining protocol.In the present study, the secondary antibody was preab-sorbed against rat IgG, and the immunoreaction productin the control section was minimal (Fig. 8A). There was amodest amount of labeling at the edge of the section and inthe choroid plexus. This result was similar to that of

Fig. 3. The developmental regulation of specific astrocyte andoligodendrocyte marker proteins were examined by using immunoblotanalysis. Whole brain protein samples of postnatal day 0 (P0; lanesA,C,E) and P30 (lanes B,D,F) rats were blotted and probed withantibodies to glial fibrillary acidic protein (GFAP; lanes A and B),vimentin (lanes C and D), and myelin basic protein (lanes E and F).The expression of GFAP is dramatically upregulated from P0 to P30,whereas vimentin expression is greatly reduced. Myelin basic proteinshows no expression at P0 but is highly expressed at P30. This figure isan untouched digital reproduction generated by using Adobe Photo-shop 3.0.


additional controls when substituting a nonimmune mono-clonal antibody into the protocol (data not shown). Whenthe section stained with the AMP1 antibody was comparedwith control sections, a substantial amount of antibody-specific staining was observed. The most heavily labeledstructures were the choroid plexus and the ependymalcells lining the lateral ventricle. Relatively high AMP1immunoreactivity was also observed at the glia limitans.This labeling appeared to be specific to the AMP1 antibodybecause the individual cellular processes could be ob-served, and this type of labeling was not seen in the controlsection. Along with these high levels of reaction product,there was also a modest amount of diffuse labeling in theparenchyma of the brain. The levels of AMP1 immunoreac-tivity in the parenchyma slowly increased over the nextseveral weeks.

Although the overall levels of AMP1 immunoreactivityincreased between P7 and P14, the general pattern ofexpression was similar. Therefore, we describe only theAMP1 immunoreactivity observed at P14 (Fig. 8C). Insections stained with the AMP1 antibody, there was an

increase in immunoreactivity throughout the brain. Thehighest density of staining was seen in the glia limitans,choroid plexus, and ependymal lining of the ventricularsystem. In these structures, there were indications thatTAPA was concentrated at specific regions of the cells. Thebest example of this was the choroid plexus. At highmagnifications, the immunoreaction product was concen-trated at regions of cell–cell contact (Fig. 9A), reminiscentof the staining pattern observed in confluent cultures oftype 1 astrocytes. This labeling outlined the individualchoroidal cells, giving the appearance of a tiled mosaic. Asimilar type of cellular distribution and patterning wasalso seen in the ependymal lining of the ventricular system(data not shown). Finally, an increase in immunoreactivitywas observed surrounding all blood vessels (Fig. 9B). Inthese young animals, immunoreaction product outlinedsmall cell bodies in the gray matter and white matter.

Distribution of TAPA in the adult brain

When tissue sections of the adult brain were stainedwith the AMP1 antibody, a reticulated pattern of labeling

Fig. 4. A–D: The pattern ofAMP1 labeling is illustrated in photomicro-graphs of mixed glial cultures. The cultures were stained withAMP1 (A,C).A and B are taken from the same region, with A representing thedistribution of AMP1 and B illustrating the distribution of astrocytesmarked with anti-glial fibrillary acidic protein. C and D are taken from the

same region. This culture was counterstained with the CE1 antibody (D) toidentify oligodendrocytes. The AMP1 antibody labels the external surfaceof both astrocytes and oligodendrocytes because the antibody was appliedto cultures of living glia. All photomicrographs taken at the same magnifi-cation. Scale bar 5 50 µm.


was seen throughout the tissue, with clear evidence ofspecific concentrations of immunoreaction product(Fig. 8D). All regions with elevated levels of immunolabelwere associated with glial structures. At the glia limitans,there was an elevation of labeling just beneath the pialsurface. High levels of AMP1 labeling were also observedat the ventricular surface, where TAPA was seen toconcentrate at regions of cellular contact between andbeneath the ependymal cells. Similar to the ependyma, thechoroid plexus also displayed intense AMP1 immunoreac-tivity. If the choroidal cells were examined face on, thepattern of labeling exquisitely marked the junctionalregions between cells, leaving a mosaic pattern of staining.In the gray matter and white matter of the adult brain, itwas difficult to identify the types of cells carrying theimmunoreaction product. In the gray matter, a denseirregular reticulated pattern of labeling was seen withlittle indication of neuronal labeling. The pattern observedin the white matter differed from that in the gray matter,with a regular linear array to the staining pattern. Theorientation of this linear array mimicked the direction ofaxonal pathways within the section of white matter. Thepattern of labeling in the adult brain was consistent with ageneralized staining of glial processes.

When sections through the adult spinal cord werestained with AMP1 (Fig. 10C), the general pattern ofimmunoreactivity was similar to that observed in sectionsstained with the astrocyte marker GFAP (Fig. 10A). The

intensity of labeling in the gray matter was higher thanthat of the white matter, where bundles of axons wererunning up and down the spinal cord. Similar to the brain,there was a general increase in labeling at the glialimitans. There were also elevated levels of GFAP andAMP1 immunoreactivity around the central canal. Themost intriguing similarity between these two sections wasthe staining of the trabecular network of astrocytic pro-cesses by anti-GFAP antibodies and the pattern in theAMP1-stained sections. This was particularly evident athigh magnifications, where the pattern of GFAP-positiveprocesses (Fig. 10B) was very similar to that found in theAMP1-stained section (Fig. 10D). Interestingly, there wereno indications that the compact myelin was labeled withthe AMP1 antibody in these sections of the spinal cord.This appeared to contradict data from tissue culturestudies, in which the AMP1 antibody stained both astro-cytes and oligodendrocytes.

To provide an independent means of assessing thedistribution of TAPA, samples of total brain, gray matter,white matter, and myelin were analyzed by using aquantitative immunoblot method. This approach demon-strated that TAPA was found at approximately equal levelsin gray matter, white matter, and myelin (data not shown).Therefore, TAPA is in compact myelin and appears to beexpressed by all glial cells. Although there were a numberof possible explanations for the lack of immunostaining ofcompact myelin, the most parsimonious explanation was

Fig. 5. The pattern of target of the antiproliferative antibody(TAPA) immunoreactivity in the postnatal day 14 cerebellum isillustrated. A,B are taken from a double-stained section, with Ashowing the distribution of glial fibrillary acidic protein (GFAP) and Bthe localization of TAPA. Notice that the linear arrays of TAPA in the

molecular layer (arrowhead) is similar to that of the GFAP-stainedBergman glial processes (arrowhead). The pial surface is indicated byarrows. The Purkinje cells are stained in C (arrows). The pattern ofthese neuronal processes is very different from that of TAPA. Allphotomicrographs are at the same magnification. Scale bar 5 50 µm.


that the extracellular epitope was hidden within the wrapsof the compact myelin.

DISCUSSION

The present study examines the expression of TAPA inthe developing rat brain and spinal cord. Our immunoblotanalysis shows three distinct phases in the regulation ofTAPA within the brain. Before the second postnatal week,the levels of TAPA are relatively low. During the secondpostnatal week, there is a dramatic upregulation of theprotein. During the last phase, the levels of TAPA continueto increase gradually until adult levels are reached. Inaddition to the overall change in the levels of TAPA, thereis a striking alteration in the distribution of the protein.The relatively low levels of TAPA observed in the embry-onic brain are associated with the glia limitans, theependymal lining of the ventricular system, and the cho-roid plexus. This pattern of TAPA expression remainsrelatively constant from E18 through the first postnatalweek. During this developmental period, most of theneurons within the brain are born and are entering theirfinal stages of development (Berry and Rogers, 1965). Thelow levels of TAPA and the lack of detectable neuronallabeling in the brain or spinal cord demonstrate that TAPA

is not directly involved in this phase of neuronal develop-ment. The labeling observed at the glia limitans, theependymal lining of the ventricle, and the choroid plexusindicates that TAPA is specifically involved with these glialcells.

During the second postnatal week, there is a dramaticincrease in the overall levels of TAPA. The relative levels ofTAPA go from 15% of adult at P7 to 70% of the adult byP14. As observed at earlier stages of development, theependymal cells, the choroid plexus, and glia limitans arelabeled by the antibody. The predominant change in thedistribution of the protein occurs within the parenchymaof the brain and spinal cord. A fine reticulated pattern ofimmunoreaction product is found throughout the tissuewith labeling of small cell bodies. This labeling of small cellbodies may reflect the expression of TAPA by both matur-ing astrocytes and oligodendrocytes. The second postnatalweek is a very interesting developmental period, withwell-characterized changes in expression of glial proteins.One indicative change in maturing astrocytes is the down-regulation of vimentin as GFAP expression increases(Dahl, 1981). It is also during this period that astrocytesacquire the ability to become reactive and to form a glialscar (Berry et al., 1983). Oligodendrocytes are also bornduring this period and begin to form myelin. These cells

Fig. 6. The pattern of target of the antiproliferative antibody(TAPA) mRNA expression is shown by using in situ hybridization.A,B are low magnification photomicrographs of the hippocampus fromadjacent sections of the adult rat brain. C,D are higher magnificationphotomicrographs taken from the CA3/alveus region. The sectionillustrated in A and C was probed with the sense riboprobe, and thesection shown in B and D was labeled with the antisense riboprobe. At

the low magnification, the general pattern of labeling can be observed.Notice that the choroid plexus (double arrow) and ependymal lining ofthe ventricles (single arrow) are heavily labeled. At a high magnifica-tion, many cell bodies are labeled with the antisense probe, and thereis a lack of labeling in the pyramidal cell layer (arrowheads). A,B andC,D are at the same magnifications. Scale bars 5 500 µm in A,B, 100µm in C,D.


express a number of unique and well-characterized pro-teins that are involved in the formation of myelin (Raff etal., 1978, 1979; Reynolds and Wilkin, 1988; Hardy andReynolds, 1991; Miller, 1996). One of these proteins,myelin basic protein, is expressed by mature oligodendro-cytes and compact myelin. We have found that TAPA levelsincrease dramatically at this time, suggesting that it isexpressed by both astrocytes and oligodendrocytes. Thisfinding is supported by the evidence from tissue culture,where both astrocytes and oligodendrocytes are labeled bythe AMP1 antibody.

After the second postnatal week, there is a gradualincrease in the levels of TAPA. During this period, thelevels of TAPA within the parenchyma of the brain appearto increase. In the white matter tracts, a fine linear arrayof immunolabeling is observed, and there is very littleindication of staining within the compact myelin. One ofthe key developmental events occurring during these lastphases of development is the myelination of axons byoligodendrocytes. Because immunoreactivity in compactmyelin was not observed, biochemical methods were usedto assess the levels of TAPA in the myelin sheaths. Thisanalysis of myelin isolated from the adult brain demon-strates that TAPA is present in myelin at levels equivalentto that of the white matter or gray matter. To address thetime frame of TAPA expression relative to myelin produc-tion, the temporal expression of myelin basic protein was

defined. At P7 myelin basic protein was not detectable byimmunoblot methods, and only moderate levels wereobserved by P14. At the next time point examined, P30,near adult levels are observed. These results are consis-tent with the observations of Hardy and Reynolds (1991)and LeVine and Goldman (1988) who demonstrated ma-ture oligodendrocytes around P10 but very few at P7. Thegradual increase in TAPA expression in the later phases ofdevelopment appears to be attributed to the late matura-tion of oligodendrocytes and compact myelin.

Cells expressing TAPA

Within the brain and spinal cord, a variety of cell typesexpress TAPA. The first cells to express it are ependymalcells and choroidal cells. These cells are clearly labeled atE18 and remain labeled in the adult animal. The dramaticupregulation of TAPA during the second postnatal weekappears to be associated with the expression of this proteinby astrocytes and oligodendrocytes. This is substantiatedby tissue culture experiments in which both astrocytes andoligodendrocytes were labeled by an antibody directedagainst TAPA. The last major class of glia are microglia(Flaris et al., 1993). In the present study, we have notmade any attempt to examine the expression of TAPA inthese cells. Finally, the potential expression of TAPA byneurons is more difficult to assess. In the embryonic andearly postnatal brain, neurons do not appear to express

Fig. 7. The pattern of target of the antiproliferative antibody(TAPA) immunoreactivity (A,C) and glial fibrillary acidic protein(GFAP; B,D) in the hippocampus is illustrated. At a low magnification,the overall pattern of TAPA (A) and GFAP (B) immunoreactivity is

similar, with the obvious exception that TAPA labels the ependyma(arrow in C) and GFAP does not (arrow in D). A,B and C,D are at thesame magnifications. Scale bars 5 500 µm in A,B, 100 µm in C,D.


Fig. 8. The distribution of AMP1 immunoreactivity in the rat brainis illustrated at three different developmental ages. A,B: Sections froma postnatal day (P) 0 rat brain. C: Section from a P14 rat brain.D: Coronal section from an adult brain. A is a control section stained ina manner identical to that of B with the exception that the primaryantibody was omitted. Notice that there is minimal staining with thesecondary antibody alone. In B, the immunolabeling pattern of the

AMP1 antibody is shown. The most dramatic labeling is seen in thechoroid plexus (arrowheads) and glia limitans (arrows). As the brainmatures, the levels of immunoreactivity are dramatically upregulated,as can be seen in the section from the P14 brain (C). The highest levelsare found in the adult (D). All photomicrographs are taken at the samemagnification. Scale bar 5 500 µm.

TAPA, for there is no labeling of the parenchyma of thebrain. Once the fine reticulated pattern of labeling appearsin the gray matter and white matter, it is very difficult todefine which of the cell types are labeled by the AMP1antibody. To determine whether neurons in the adult brainexpress TAPA, we turned to in situ hybridization. In theseexperiments, the only cells that were labeled by theantisense TAPA probe had small cell bodies, and no clearneuronal labeling was observed. This lack of neuronallabeling was particularly clear for the pyramidal cells ofthe hippocampus and the cerebral cortex, where the largecell bodies could be seen, and these cells were not labeled.Even though we have not observed any indication ofneuronal labeling, we cannot exclude the possibility thatsome population of neuron expresses TAPA. Taken to-gether, these data demonstrate that TAPA is expressed byglial cells during a postnatal period of astrocyte andoligodendrocyte maturation.

Astrocyte maturation

Astrocytes have many different functional roles in theCNS. During the development of the brain and spinal cord,astrocytes are involved in the migration of neurons fromgerminal zones to their final locations (Hatten, 1990;Anton et al., 1996). In addition, interactions betweenastrocytes and growth cones are thought to be critical tothe formation of neuronal pathways (Silver and Rut-ishauser, 1984). Later in development, specific sets ofastrocytes in boundary regions express proteins believedto separate functional units of the CNS (Steindler et al.,1990; Geisert and Bidanset, 1993). As development contin-ues, the astrocytes appear to take over a role of maintain-ing a homeostatic environment within the CNS and form-

ing protective barriers (Walz and Hertz, 1982; Ballanyi etal., 1987). Astrocytic endfeet at the edge of the brainprovide one of the key structural components of the glialimitans. Astrocyte contact with endothelial cells inducesthe formation of the blood–brain barrier. Astrocytes aid inthe regulation of ionic and molecular compounds in theextracellular fluids through specific uptake mechanisms.The astrocytic barriers are located at each point of contactbetween the CNS and the outside world. In addition,during the final stages of development, astrocytes arebelieved to be directly involved in the stabilization of thestructure of the CNS and synaptic connectivity patterns. Itis during this final stage of development that astrocytesacquire the ability to respond to injury and become reac-tive (Berry et al., 1983). Part of this response may be theexpression of major histocompatibility II proteins and theacquisition of macrophagelike features (Fontana et al.,1984; Takiguchi and Frelinger, 1986), which can associatewith human TAPA-1 in the plane of the cell membrane(Secrist et al., 1996). During this period of development,there is a tight correlation between the upregulation ofTAPA at the site of injury and the ability of astrocytes tobecome reactive, as judged by GFAP staining (Geisert,unpublished observations).

Role of TAPA

The present paper focuses on the expression and the roleof the glial membrane protein TAPA during development.TAPA is a member of the tetramembrane spanning familyof proteins, containing four transmembrane domains withone major extracellular loop and intracellular C- andN-terminal regions (Oren et al., 1990; Takahashi et al.,1990; Virtaneva et al., 1993). All of these proteins (CD9,

Fig. 9. The pattern of immunolabeling in the choroid plexus (A)and the junction between white and gray matters (B) can be seen inthese high magnification photomicrographs taken from the postnatalday 14 brain. The choroid plexus illustrates the high levels of target ofthe antiproliferative antibody immunoreactivity at the sites of cell–

cell contact (arrowheads). Note the difference in the staining patternat the junction between the gray matter and white matter, along withthe staining of small cell bodies (arrows) with AMP1 (B). Bothphotomicrographs are taken at the same magnification. Scale bar 550 µm.


CD37, CD53, CD63, TAPA-1, CO-029, R2, KAI1, and latebloomer) have considerable sequence homology at theamino acid level (Jennings et al., 1990; Oren et al., 1990;Kaprielian and Patterson, 1993; Yatomi et al., 1993; Donget al., 1995; Kaprielian et al., 1995; Kopczynski et al.,1996). The only other member that has been found in themammalian nervous system is CD9. It is expressed bySchwann cells in the peripheral nervous system (Kapri-elian and Patterson, 1993) and appears to be expressed byoligodendrocytes in the CNS (Deissler et al., 1996; Naka-mura et al., 1996).

In several cases, the molecular interactions of the tetra-membrane spanning family members are defined. On theone hand, some family members interact with traditionaladhesion molecules. For example, CD9 is associated withb1 integrins (Slupsky et al., 1989; Masellis-Smith andShaw, 1994). On the other hand, some of the tetramem-brane spanning family members are associated with eitherkinase or phosphatase activity. These associations appearto link cell adhesion to second-messenger systems. These

molecular cascades affect cell behaviors such as mitoticactivity and cell adhesion (Nakamura et al., 1995). Forexample, during platelet activation (Jennings et al., 1990;Yatomi et al., 1993), there is an increase in phosphoryla-tion that can be replicated by the binding of the CD9antibody. Similar effects are observed with TAPA-1, whereantibody binding activates a tyrosine kinase (Schick et al.,1993). Finally, late bloomer appears to be associated withadhesion at the neuromuscular junction and phosphataseactivity (Kopczynski et al., 1996).

Like other members of this family of proteins, ananalysis of the deduced structure of TAPA (Fig. 11) showsthat a large part of the molecule is found within the cellmembrane, with four transmembrane domains accountingfor approximately 40% of its structure. It also exhibits alarge and small extracellular loop. Assuming that thecystines at amino acids 156 and 190 of the large extracellu-lar loop are linked by a disulfide bond, both loops would beclosely associated with the cell membrane. Furthermore,based on a secondary structural analysis by using predic-

Fig. 10. The distribution of AMP1 immunoreactivity in the adultspinal cord is illustrated in a series of low and high magnificationphotomicrographs. C,D: High levels of AMP1 immunoreactivity areobserved in the spinal cord. A,B: In a similar section stained for glialfibrillary acidic protein (GFAP), the distribution of astrocytes in the

spinal cord is illustrated. As in cortex, the AMP1 immunoreactivity inwhite matter is lower than that in the gray matter (C). Note thesimilarity in the staining patterns between the anti-GFAP- andAMP1-stained sections (arrowheads). A,C, and B,D are at the samemagnifications. Scale bars 5 500 mm in A,C, 250 mm in B,D.


tions from Chou and Fasman (1974) and Garnier et al.(1978), the loop forms an alpha helix. Because each aminoacid in an alpha helix represents a distance of 1.5 A, themaximum distance the large loop can extend from thesurface of the cell is 40.5 A. Even if no disulfide bonds areformed, the maximum distance the second loop can extendfrom the surface of the cell is 64.5 A. Based on X-raycrystallographic studies of N-cadherin (Shapiro et al.,1995), a cell adhesion molecule, the extracellular space isapproximately 290 A from membrane to membrane. Thus,the two extracellular domains of the molecule appear to betoo small for direct interactions across the extracellularspace.

Antibodies directed against the human hematolymphoidcell protein TAPA-1 cause an increase in adhesion betweenthese lymphoid cells and a depression in their mitoticactivity (Oren et al., 1990). This type of regulation of cellbehavior is a common theme for members of the tetramem-brane spanning family of proteins. For example, antibod-ies binding to CD9 enhance cell–cell adhesion (Forsyth,1991; Masellis-Smith and Shaw, 1994) and can affect themobility and invasiveness of tumor cells (Miyake et al.,1991). In the nervous system, the migratory behavior ofSchwann cells is enhanced with the application of antibod-ies directed against CD9 (Anton et al., 1995). The sameantibody enhances the mitotic rate of the cultured Schwanncells (Hadjiargyrou and Patterson, 1995). Another familymember, KAI1, is the metastatic suppressor for prostatecancer (Dong et al., 1995). Without this protein, theprostate cells cannot form stable cell contacts and are

metastatic. When the levels of KAI1 are elevated by genetransfection, there is a dramatic decrease in the metastaticbehavior of these cells. This theme of stabilizing cellularcontacts is also demonstrated by the most recently identi-fied member of the tetramembrane spanning family, latebloomer (Kopczynski et al., 1996). The late bloomer proteinis involved in stabilizing the synaptic contact betweenmotor neurons and muscles. Without this protein, thegrowing axon does not appear to be capable of docking withthe muscle fiber. Thus, some members of the tetramem-brane spanning family are intimately involved in theformation and maintenance of stable cell contacts, andTAPA appears to play a similar role in glial cells.

CONCLUSION

There is a general increase in the level of TAPA as glialcells are born and mature. This increase in the overalllevel of TAPA is demonstrated by our immunoblot analysis;however, this type of analysis could not demonstrate thecellular localization of TAPA. Sections from the developingbrain stained for TAPA demonstrate a clear sequence ofcellular labeling. The cells of the ependyma and choroidplexus are the first to express TAPA, followed by astrocytesand oligodendrocytes. The general increase in TAPAexpres-sion occurs at a pivotal point in development, the secondpostnatal week. During this week, astrocytes and oligoden-drocytes begin to mature, the structure of the CNS isstabilized, and astrocytes acquire the ability to becomereactive. It is during this period that the largest increasein TAPA is observed, going from 15% to 70% of the adult (afivefold increase). To date, all identified members of thetetramembrane spanning family play a role in the molecu-lar cascade, leading to the stabilization of cellular con-tacts.

ACKNOWLEDGMENTS

We thank the Center for Excellence in Neuroscience forgraduate support for C.D. Sullivan. We also thank Li JuanYang, M.D., and Allison Stewart for their technical assis-tance.

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Expression of rat target of the antiproliferative antibody (TAPA) in the developing brain

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