Proc. Natl. Acad. Sci. USAVol. 86, pp. 9337-9341, December 1989Developmental Biology

Unusual stage-specific embryonic antigen (TEC-4) defined by amonoclonal antibody to embryonal carcinoma cells defective inthe expression of embryoglycan

(cell surface/mouse embryo/monoclonal antibody/retinoic acid)

PETR DRABER, JINDAICH NOSEK, AND ZORA POKORNADepartment of Developmental Genetics, Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Videfiskd 1083, 142 20 Prague, Czechoslovakia

Communicated by Salome G. Waelsch, August 28, 1989

ABSTRACT Most developmentally regulated epitopesidentified on embryonal carcinoma cells and murine preim-plantation embryos are associated with a glycoprotein-boundlarge glycan called embryoglycan. To prepare monoclonalantibodies recognizing other, less immunogenic stage-specificembryonic epitopes, we used embryoglycan-negative embryo-nal carcinoma cells P19XT.1.1 as immunogen. One monoclonalantibody prepared by this strategy was found to react specif-ically with mouse embryonal carcinoma and embryo-derivedstem cell lines. The target epitope, TEC-4, was found to beexpressed on eggs and two-cell embryos but was undetectableon later stages of mouse embryos and adult mouse tissues.NaDodSO4/PAGE of immunoaffinity-isolated antigen revealedthat TEC-4 epitope is associated with glycoproteins of apparentMr 120,000 and 240,000. The epitope was resistant to oxidationby sodium periodate and to digestion by endoglycosidase F butwas sensitive to treatment with protein-denaturing agents andproteases, which suggested that the epitope is located in theprotein moiety of the molecule. In the course of retinoicacid-induced differentiation of embryonal carcinoma cells theepitope disappeared before the onset of morphological differ-entiation. The combined data indicate that TEC-4 is an unusualstage-specific embryonic antigen that may be amenable todirect genetic analysis.

The processes of embryonic development seem to involvesome stage- and/or tissue-specific cell-surface molecules thattrigger or reflect particular development commitments andplay a functional role in their maintenance. Information onsuch molecules is scanty and often relies on availability ofmonoclonal antibodies that can be used as highly specificimmunological probes for the identification, isolation, ormasking of these developmentally interesting molecules orfor manipulating their expression. Antibodies recognizingstage-specific embryonic antigens (SSEAs) have been raisedby immunization with early embryonic cells (1) and particu-larly with murine embryonal carcinoma (EC) cells (2) becausethese cells share many properties, including the antigenicmakeup, with mouse preimplantion-stage embryos and pro-vide a convenient source of immunizing agents (3).

Ideally, analysis of SSEAs should include not only exam-ination oftheir biochemical properties and patterns of expres-sion but also molecular characterization of the correspondinggenes and their transcriptional products. However, molecu-lar analysis of most ofthe hitherto identified SSEAs would bedifficult because the developmentally regulated epitopesidentified by monoclonal antibodies are located in the car-bohydrate portion of the surface glycoconjugates. Perhapsthe best characterized carbohydrate epitope is that of SSEA-1 (2), which is localized in a glycoprotein-bound large glycan

called embryoglycan (4). Embryoglycan also carries a num-ber of other stage-specific embryonic epitopes such asECMA 2 and ECMA 3 (5), NL-9, Thy-22, and HL-5 (6),TEC-2 (7), TEC-5 (8), and sialyl Lex (9). When whole EC cellsare used as immunogen, developmentally regulated carbo-hydrate epitopes generally behave as immunodominant, andcarbohydrate-specific antibodies are obtained with relativelyhigh frequency. To overcome this problem, we have beenexploring an alternative strategy for achieving the centralgoal of relating SSEAs to particular gene products. Here, werely upon the production of antibodies after immunizationwith EC cells genetically deprived of immunodominant car-bohydrate structures. Ideally, such cells should have prop-erties of EC cells but should not express embryoglycan.Mutants defective in the expression of SSEA-1 have been

isolated in this laboratory from mutagenized P19 EC cells (10)by a single-step selection technique using monoclonal anti-body TEC-O1 conjugated with the plant toxin ricin (11). Ofthree independently isolated mutant celiies, one apparentlylacked embryoglycan and carbohydrate sttures typical ofearly embryonic and EC cells, but the cells still exhibitedmany properties of EC cells, including morphology andability to differentiate in vitro. In the present report wedescribe an unusual SSEA, TEC-4, which was identified bya monoclonal antibody raised against the embryoglycan-defective EC cells. The TEC-4 epitope seems to be localizedin the protein portion of a developmentally regulated glyco-protein.

MATERIALS AND METHODSCell Cultures and Induction of Differentiation. The origin of

most cell lines used in this study and the culture conditionshave been described (7, 12). P19XT.1.1 is an embryoglycan-defective subclone derived from P19X1 (11). RAC65 is asubclone of P19 cells that does not differentiate in vitro (13).NTERA-2 cl.D1 (14) and N2102Ep cl.2A6 (15) were obtainedfrom P. Goodfellow (Imperial Cancer Research Fund, Lon-don). Embryo-derived stem (ES) cell lines were isolated bya modified procedure (16, 17) by S. Gregorova (ES3Ph/4 andES5Ph/2) and M. Mickova (ES7Ph/3) of this department.TEC-04 Antibody. Lewis rats were immunized at 2-week

intervals by s.c. and i.p. injection of 2-5 x 107 P19XT.1.1cells. The rats were tail-bled 7 days after each injection, andsera were tested for reactivity on EC cells by indirect RIA(18). A rat that showed a high antibody response was boosted4 days before cell fusion. Its spleen cells were fused withX63-Ag8.653 myeloma cells, and hybridoma cells were iso-lated as described (19, 20). TEC-04-containing ascites wasprepared by i.p. injection of hybridoma cells into pristane(2,6,10,14-tetramethylpentadecane) primed nude mice. TEC-

Abbreviations: EC, embryonal carcinoma; ES, embryo-derivedstem; SSEA, stage-specific embryonic antigen.

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04 IgG was purified from ascites fluid by precipitation with50% saturated ammonium sulfate, followed by ion-exchangechromatography using DEAE-Sephadex A-50 (Pharmacia).Purification of TEC-01, TEC-02, and OX-7 antibodies, spe-cific for the Gal-(81-+4)-[Fuc(al-+3)] N-acetylglucosamine(GlcNAc) sequence, GalNAc-(,81--+4)-Gal(1-+4) sequence,and Thy-1.1 antigen, respectively, has been described (7, 21,22).

Indirect Immunofluorescence and Cytotoxicity Test. Unfer-tilized and fertilized eggs and embryos were isolated fromBALB/c mice, and the zonae pellucidae were removed byacid Tyrode's solution (8). Zona pellucida-free eggs andembryos and EC cells were exposed to TEC-04 antibody (50,ug/ml in cell culture medium) for 30 min at room tempera-ture, then washed and treated with rabbit IgG anti-TEC-04IgG, followed by swine IgG anti-rabbit IgG conjugated withfluorescein isothiocyanate. This three-step procedure wasmore sensitive than the two-step procedure. The cells wereanalyzed as described (8) or by using a fluoresence-activatedcell sorter (FACS 440, Becton Dickinson: analysis performedby P. Hausner, Faculty of General Medicine, Prague). TEC-01 antibody binding was detected with swine IgG anti-mouseIgM conjugated with fluorescein isothiocyanate. Cytotoxic-ity was determined by using rabbit serum as the source ofcomplement. The cells (2 x 101) were incubated in 100 Ala ofTEC-04-containing ascites, diluted in culture medium for 30min at 40C, then washed, resuspended in 50 ,l of rabbitserum, diluted 1:5 in serum-free medium, and incubated for40 min at 370C. The percentage of lysed cells was determinedusing trypan blue staining. In antibody alone and complementalone the proportion of lysed cells did not exceed 20%.

Radioantibody-Binding Assays. Isolated monoclonal anti-bodies were iodinated by the chloramine-T method (23). Thespecific activity of iodinated antibodies was 1-2 x 107 cpm/,g, and the preparation contained 60-88% bindable anti-body. Direct RIA, absorption assay, construction of satura-tion curves, and calculation of Scatchard plots were per-formed as described (8, 24). Competitive assay was employedfor monitoring the presence of TEC-4 antigen in cell lysates.The sample to be analyzed (20 ,ul) was mixed with 30 Ala of1251-labeled TEC-04 (105 cpm) and incubated for 6 hr at 4°C.Glutaraldehyde-fixed P19X1 cells (5 x 105 in 50 ,ul) were thenadded, and incubation was continued for 90 min. The incu-bation mixture was then layered over 1 ml of 10% bovineserum albumin in 0.01 M Na2HPO4/0.15 M NaCl, pH 7.4(Pi/NaCI) in another tube and centrifuged at 500 x g for 10min. The tube was frozen, the tip containing cell-boundantibody was cut off, and the radioactivity in the tip wasmeasured.

Isolation and Characterization of TEC4 Antigen. TEC-04antibody was immobilized on CNBr-activated Sepharose 4B(Pharmacia). P19X1 cells (109) were solubilized in lysis buffer(150mM NaCI/5 mM EDTA/50 mM Tris HCl, pH 8.0/0.02%NaN3/1 mM phenylmethylsulfonyl fluoride) containing 0.5%Nonidet P-40 for 30 min at 0°C and then centrifuged to removenuclei. The supernatant was then centrifuged at 100,000 x gfor 30 min and applied to the TEC-04-Sepharose 4B column(3 ml). The column was washed with 20 ml of lysis buffer, andthe antigen was eluted with 0.5% sodium deoxycholate in 0.1M glycine-NaOH buffer, pH 11.5. Fractions (200 ,ul) werecollected and neutralized by 0.3 M NaH2PO4. Fractionscontaining TEC-4 antigen were pooled, dialyzed overnightagainst 50 mM Tris'HCI, pH 8.1, containing sodium deoxy-cholate and concentrated by ultrafiltration. TEC-4 antigenwas iodinated by the chloramine-T method and purified bygel filtration on Sephadex G-150 (Pharmacia) in 10 mMTris'HCI, pH 8.2, containing 0.1% sodium deoxycholate. Insome experiments, TEC-4 antigen was isolated from lysatesof cells labeled by lactoperoxidase-catalyzed iodination (25)or from cells labeled metabolically with [6-3H]galactose (7).

Portions of the radiolabeled TEC-4 antigen (-70,000 cpm)were incubated for 1 hr at 370C with (i) 40 pug of Pronase in50 mM Tris HCl, pH 8.0/10 mM CaCl2; (ii) 20 1Lg of pepsinin 100 mM sodium acetate, pH 4.5; or (iii) 10 p.g of papain in0.01 M Na2HPO4/0.15M NaCl (pH 7.4)/2mM EDTA/10mML-cysteine. Other aliquots of unlabeled or 125I-labeled TEC-4antigen were treated with endoglycosidase F (BoehringerMannheim) or endoglycosidase H (Sigma) as described (26).

Gel Electrophoresis and Immunoblot Analysis. NaDodSO4/PAGE was performed as described (27). Proteins from un-stained gels were transferred to nitrocellulose paper (28), andthe remaining protein-binding sites were blocked with 5%low-fat milk in 0.01 M Na2HPO4/0.15 M NaCl (pH 7.4). Theblots were incubated with 1251I-labeled TEC-04 antibody in0.01 M Na2HPO4/0.15 M NaCl/0.05% Tween 20 and pro-cessed for autoradiography. Alternatively, blots were incu-bated with TEC-04 antibody and then a solution of porcineanti-mouse IgG conjugated to peroxidase was added. Bandsof antigen were visualized by H202/aminoethylcarbazolechromogenic substrate solution (29).

Solid-Phase Assay. Antibodies (1 ,ug in 50 ,ul of 50 mMNaHCO3, pH 8.6) were immobilized to the high-bindingcapacity microwells (Nunc). After overnight incubation, theremaining binding sites were blocked by 0.01 M Na2HPO4/0.15 M NaCl (pH 7.4) supplemented with 0.5% bovine serumalbumin for 1 hr at 37°C, and the wells were washed with 0.01M Na2HPO4/0.15 M NaCl (pH 7.4)/0.05% Tween 20. Toeach well 50 ,ul of 1251I-labeled TEC-4 antigen (30,000 cpm) in0.01 M Na2HPO4/0.15M NaCl (pH 7.4)/0.05% Tween 20 wasadded, and the wells were incubated for 2 hr at 4°C. Afterwashing, the wells were assayed for radioactivity in a ycounter. All assays were performed in triplicate; the SD forall values above 200 cpm was <10%.

RESULTSTEC-04 Antibody and Its Reactivity with Cell Lines. Injec-

tion of embryoglycan-negative P19XT.1.1 cells into Lewisrats induced antibody responses detectable by the indirectRIA with antiserum diluted up to 1:105. There was nosignificant difference when the antisera were tested on em-bryoglycan-negative or parental P19X1 cells. Monoclonalantibody TEC-04 was initially selected for its reactivity withP19XT.1.1 cells and nonreactivity with PYS-2 cells. Asrevealed by NaDodSO4/PAGE (data not shown), the anti-body was of IgG class. Extensive testing against a panel ofcell lines showed that the target epitope, named TEC-4, isexpressed not only on cells used for immunization but also onparental P19X1 cells and various derivatives of P19 EC cells(Table 1). The antibody also bound to several other EC celllines and to all ES cell lines tested. Nonreactive cells includednulli-potent EC cells F9, parietal endoderm cells PYS-2, andSTO fibroblasts. The antibody also did not react with severalother murine-transformed cell lines, Chinese hamster ovarycells, human teratocarcinoma-derived cell lines NTERA-2cl.D1 and N2102Ep cl. 2A6, and several other human celllines (data not shown). Interestingly, the antibody bound tomouse neuroblastoma cells C1300/E7 that had been shown tobe negative for the expression of other SSEAs (7). In thepresence of rabbit complement the TEC-04 antibody-con-taining ascites lysed 90% ofP19X1 cells at 1:3000 dilution and50% of the cells at 1:25,000 dilution.TEC-4 Epitope in Eggs, Embryos, and Adult Mouse Tissues.

Indirect immunofluorescence studies of eggs and preimplan-tation-stage embryos revealed that TEC-04 antibody binds tounfertilized eggs (Fig. la), fertilized eggs, and two-cell em-bryos (Fig. lb), but not to four-cell embryos or the laterstages of preimplantation embryos. Cryostat sections of6-day-old embryos also showed no detectable staining. Ab-sorption assays indicated that TEC-04 antibody does not bind

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Table 1. Reactivity of TEC-04 antibody with cell lines

Radioactivityin cell pellet*,

Cell line Description (origin) % cpm

Murine teratocarcinoma-derivedP19X1 EC (from P19) 7.5 (5.7-9.4)P19XT.1.1 EC (from P19X1) 10.9 (9.8-12.6)P19S1801A1 EC (from P19) 11.6 (8.7-14.1)P19ST.1.3 EC (from P19S18O1A1) 8.1 (7.6-8.8)P19ST.1.5 EC (from P19S1801A1) 7.6 (5.2-10.4)RAC65 EC (from P19S18RAC6) 9.7 (8.7-11.6)F9 EC (from 0TT6050) <0.2PCC3/A/1 EC (from 0TT6050) 3.2 (2.4-4.3)PCC4/Aza EC (from 01T6050) 7.9 (5.4-10.8)PYS-2 Parietal endoderm <0.2

(from 0TT6050)Murine embryo-derived

ES3Ph/4 ES (129-T/t0 x C3H/Di)tO/+ 5.6 (3.9-7.4)ES5Ph/2 ES (129-T/t0 x C3H/Di)tO/+ 6.4 (5.1-7.2)ES7Ph/3 ES (129-t0) 3.4 (2.2-5.2)

Murine transformedSTO Fibroblast (SIM) <0.2C1300/E7 Neuroblastoma 2.8 (1.8-4.4)*Cells (2.5 x 105) were incubated with I251-labeled TEC-04 antibody(105 cpm), and percentage of radioactivity bound was determined indirect RIA. Average values and ranges (in parentheses) of triplicatemeasurements from three to five independent experiments areshown.

significantly to tissue homogenates from brain, heart, skeletalmuscle, lung, liver, spleen, thymus, testes, epididymis, andstomach.TEC-4 Epitope in Differentiating P19X1 Cells. When mono-

layer cultures of P19 cells are treated with retinoic acid,

virtually all cells differentiate into fibroblast-like cells (30).Fig. 2 shows that this differentiation was accompanied byprogressive loss of TEC-4 epitope expression. In culturestreated with retinoic acid for 3 days, the binding of iodinatedTEC-04 antibody to constant number of cells was reduced to20% (Fig. 2a); this loss of reactivity was associated withdisappearance of TEC-4-positive cells as detected by theindirect immunofluorescence assay (Fig. 2b). It should benoted that the disappearance of the TEC-4 epitope signifi-cantly preceded the decrease in expression of SSEA-1 asdetected by TEC-01 antibody. In fact, after 48 hr in thepresence of retinoic acid, when the cells did not yet exhibitsignificant changes in morphology, the staining of individualcells with TEC-04 was significantly reduced (Fig. 3). Thesame pattern ofchanges in the TEC-4 epitope expression wasseen in retinoic acid-treated cultures of P19XT.1.1 cells (datanot shown).Number of TEC-4 Epitopes. The number of TEC-4

epitopes, as estimated from Scatchard analysis of data fromtwo independent saturation assays, was 2.0 (± 0.2) x i0 percell. This value corresponds to =4 ,g ofprotein ofMr 120,000(see below) per 108 cells.Inmmunochemical Characterization ofTEC-4 Antigen. TEC-

4 antigen was isolated from lysates of P19X1 cells by immu-noaffinity chromatography. After iodination, the antigen wasrepurified and subjected to NaDodSO4/PAGE. Under reduc-ing conditions, TEC-4 migrated as a single band with appar-ent Mr 120,000 (Fig. 4 lane a). A glycoprotein of the sameapparent Mr was also isolated by immunoaffinity chromatog-raphy from lysates of cells labeled by lactoperoxidase-catalyzed iodination (Fig. 4 lane b) or labeled metabolicallywith [6-H]galactose (Fig 4 lane c). Some preparations alsocontained a band of apparent Mr 240,000 (Fig. 4 lane a).Microgram quantities of TEC-4 antigen isolated were run onparallel gradient gels and subsequently either stained withCoomassie blue or blotted onto nitrocellulose and visualizedimmunoenzymatically. In reduced samples TEC-4 wasmostly present in the lesser form of apparent Mr 120,000,whereas the band corresponding to apparent Mr 240,000contained less protein (Fig. 5 lanes a and b). In unreducedsamples, bands corresponding to both forms were morediffuse and migrated either with the same velocity (boiledsamples; Fig. 5 lane c) or with somewhat higher velocity(unboiled samples; Fig. 5 lane d). TEC-04 antibody reactedstrongly with both forms of the antigen blotted onto nitro-

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O 1 2 3 4 5 0 1 2 3 4 5Retinoic acid treatment (days)

FIG. 1. Indirect immunofluorescence staining of unfertilized egg(a) and two-cell-stage embryo (b) with TEC-04 antibody. Note thatin b the morula-stage embryo is negative. Photos were taken usingUV illumination (Left) or bright-field illumination (Right) (a, x250;b, x180).

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FIG. 2. Binding of TEC-04 antibody to P19X1 cells in the courseof retinoic acid-induced differentiation. The cells were incubatedwith 0.5 AM retinoic acid for the number of days indicated, har-vested, and tested in direct RIA (a) or indirect immunofluorescenceassay (b) using TEC-04 antibody (o) or control TEC-01 antibody (o).

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FIG. 3. Flow cytofluorimetric analysis of the binding of TEC-04antibody to untreated P19X1 cells (TEC-04) or cells treated for 48 hrwith 0.5 ;LM retinoic acid (TEC-04/RA). Fluorescence intensity ofcells stained with a control, OX-7, antibody is also shown (C).

cellulose when the sample had not been boiled before elec-trophoresis (Fig. 5 lanes e and h); the reactivity with bothforms was greatly reduced (Fig. 5 lane g) or completely lost(Fig. 5 lane f) after boiling in the absence or presence ofreducing agent, respectively.These results suggested that the TEC-4 epitope is located

in the protein moiety of the molecule. Some other datalikewise pointed to the protein nature of TEC-4 epitope. Incompetitive assay, the addition ofcrude lysate ofP19X1 cellsreduced the binding of 1251-labeled TEC-04 antibody to fixedcells by 85-95%; the inhibitory activity of the lysate wasabolished by treatment with 1% NaDodSO4 and by 950C for1 min. Similar treatment had no effect on the binding ofantibodies recognizing carbohydrate epitopes (ref. 7 andunpublished data). Treatment of P19X1 cells with 50 mMsodium periodate in 0.01 M Na2HPO4/0.15 M NaCl (pH 7.4),which reduced the binding ofTEC-O1 and TEC-02 antibodiesby 99%, decreased TEC-04 antibody binding by only 20-25%.The nature of the TEC-4 antigen was further investigated

by enzyme digestion of the molecule isolated. Both [6-3H]galactose-labeled and iodinated TEC-4 molecules weredigested by Pronase, pepsin, and papain to fragments runningin the gel front; the fragments did not bind to immobilized

205-

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205 -

97 -*

66-'

a b c d e f

FIG. 4. NaDodSO4/PAGE ofTEC4 antigen purified from P19X1cells by immunoaffinity chromatography. Radiolabeled sampleswere analyzed on 1o gels (lanes a-c) or on 5-15% gradient gels(lanes d-f) and visualized by autoradiography (lanes a, b, and d-f) orfluorography (lane c). Isolated TEC4 antigen was labeled with iodineby the chloramine-T method (lane a). TEC4 antigen was isolated byaffinity chromatography from lysates of cells labeled by lactoperox-idase-catalyzed iodination (lane b) or from cells metabolically labeledwith [6-3H]galactose (lane c). 125I-labeled antigen was untreated (laned) or treated with endoglycosidase F (lane e) or endoglycosidase H(lane f). Positions of Mr markers (Mr x 10-3) run in parallel areshown at left.

220-

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a b c d e f g h i j

FIG. 5. Immunoblot analysis of TEC-4 antigen. Immunoaffinity-purified TEC-4 antigen was resolved by NaDodSO4/PAGE (5-15%gradient gel) and stained with Coomassie blue (lanes a-d) or trans-ferred to nitrocellulose and visualized by TEC-04 antibody andporcine IgG anti-mouse IgG conjugated to peroxidase (lanes e-h) orby 125I-labeled TEC-04 antibody and autoradiography (lanes i and j).The following samples of undigested TEC-4 antigen were analyzed:reduced and unboiled (lanes a and e), reduced and boiled (lanes b andf), unreduced and boiled (lanes c and g), and unreduced and unboiled(lanes d and h). Lanes i andj show reactivity ofTEC-04 antibody withboth molecular forms of native (lane i) and endoglycosidase Fdeglycosylated (lane j) TEC-4 antigen. Positions of Mr markers (MrX 10-3) are shown at left.

TEC-04 antibody in the solid-phase assay. EndoglycosidaseF reduced the apparent Mr of the iodinated antigen by20,000-30,000, whereas endoglycosidase H left the moleculeintact (Fig. 4 lanes d-f). Unlabeled TEC-4 antigen denaturedby 1% NaDodSO4 in the absence of 2-mercaptoethanol atroom temperature and digested with endoglycosidase F re-tained its reactivity with TEC-04 antibody when blotted ontonitrocellulose (Fig. 5 lanes i and j).P19X1 cells expressed both the TEC-4 epitope and the

TEC-1 (SSEA-1) carbohydrate epitope. To determine therelationship between these epitopes, the solid-phase assaywas used. The results showed that 1251-labeled TEC-4 antigenbound to the TEC-04 antibody (1880 cpm bound) but did notbind to TEC-01 antibody (190 cpm bound) or control OX-7antibody (190 cpm bound).

DISCUSSIONIn a previous paper (11) it was predicted that EC cell mutantsdefective in the expression of embryoglycan might be usefulfor the discovery, production, and characterization of mono-clonal antibodies recognizing SSEAs. Data presented in thispaper indicate that rats immunized with embryoglycan-negative P19XT.1.1 cells produced antibodies recognizingantigens expressed on both mutant and parental EC cells.One such antibody was analyzed in detail and was found torecognize a SSEA that differed in many properties from otherknown surface antigens common to mouse embryos and ECcells. Thus, the target epitope, TEC-4, was expressed on EScell lines and on a limited number ofEC cell lines, the highestexpression being on P19-derived cell lines. Furthermore,TEC-4 was found to be expressed on eggs and two-cell-stagemouse embryos, but not at later stages of preimplantationembryos and adult mouse tissues. In addition, the TEC-4epitope disappeared from the surface of P19X1 cells early inthe course of retinoic acid-induced differentiation. Finallyand more importantly the epitope was resistant to carbohy-drate-destroying agents and sensitive to protein-denaturingtreatments; this result suggested that the TEC-4 epitope islocated in the protein portion of the molecule.Immunochemical characterization indicated that the epi-

tope is carried by a glycoprotein of apparent Mr 120,000 and240,000. The glycoprotein ofMr 240,000 is apparently an S-Sbound dimer of the molecule of Mr 120,000 because it

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disappears after extensive reduction at 950C with concomi-tant increase of the intensity of a band of Mr 120,000. Theantigen is N-glycosylated and does not carry a developmen-tally regulated carbohydrate epitope recognized by TEC-01(anti-SSEA-1) antibody.The finding that the TEC-4 epitope is expressed in EC and

ES cells is important for the discussion of the developmentalstage of the embryoglycan-defective mutants we had previ-ously isolated (11). Although unlikely, the possibility re-mained that the cell lines resistant to the cytotoxic action ofa TEC-01 antibody-ricin conjugate (with phenotypes TEC-1-, 2+, 3+; TEC-1-, 2+, 3+; and TEC-1-, 2-, 3-) were celllines in which the observed changes in the expression andproperties of the embryoglycan reflected phenotypes of dif-ferentiated EC cells rather than phenotypes of mutated cells.However, the expression of TEC-4 antigen on the surface ofall of these cell lines makes such explanation very unlikelybecause TEC-4 antigen disappears from the surface early inthe course of EC cell differentiation, before the onset ofmorphological differentiation. Furthermore, when embryo-glycan-defective P19XT.1.1 cells were treated with retinoicacid, the TEC-4 epitope disappeared from the differentiatingcells within the same time interval as from the parental,embryoglycan-positive P19X1 cells.Monoclonal antibodies recognizing developmentally regu-

lated carbohydrate epitopes have proved to be invaluabletools for analysis of mouse embryogenesis and EC celldifferentiation induced by a variety of agents (31). Theavailability of TEC-04 antibody makes it possible to analyzeearly stages of this differentiation and accompanying changesin surface properties, not only in normal EC cells but also inembryoglycan-defective mutants. At present, there is noindication as to what could be the function of the TEC-4antigen. The binding ofTEC-04 antibody to P19X1 cells doesnot affect their morphology, their ability to adhere to tissue-culture surface, formation of aggregates, or differentiationinto neurons after treatment of the aggregates with retinoicacid (P.D., unpublished data). Use of polyclonal antiseraraised by immunization with affinity-purified TEC-4 antigenmay be a way of continuing analysis on the functional role ofthe TEC-4 antigen. Another approach could be isolation ofEC cell mutants defective in the expression ofTEC-4 antigenand comparison of the properties of the mutant and parentalcells.The finding that the TEC-4 epitope disappears early in the

course of retinoic acid-induced differentiation suggests thepossibility that the corresponding gene is controlled eitherdirectly by retinoic acid-mediated action or at an early pointin the differentiation program. With the recent evidence thata nuclear receptor exists for retinoic acid (32), it is nowreasonable to postulate direct control of gene expression byretinoic acid in differentiation and to determine the mecha-nism of regulation of the TEC-4 antigen expression by thismorphogen. The TEC-04 antibody may be a key to successful

cloning of the corresponding gene from expression cDNAlibraries.

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Developmental Biology: Drdber et al.

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