Proc. Nadl. Acad. Sci. USAVol. 91, pp. 8641-8645, August 1994Cell Biology

Expression of a specific marker of avian programmed cell death inboth apoptosis and necrosis

(development/immunofluorescence)

PIERRE-ALAIN FERNANDEZ*t, Rocco J. ROTELLO*t, ZEHAVA RANGINI*t, ALLISON DOUPEt,HANNES C. A. DREXLER*t, AND JUNYING YUAN*t§*Cardiovascular Research Center, Massachusetts General Hospital, 149 Thirteenth Street, Charlestown, MA 02129; tDepartment of Medicine, HarvardMedical School, Boston, MA 02115; and tDivision of Biology, California Institute of Technology, Pasadena, CA 91125

Communicated by H. Robert Horvitz, May 18, 1994 (received for review December 3, 1993)

ABSTRACT Apoptosis and necrosis are two types of celldeath with different morphologic features. We report here theisolation of a monoclonal antibody, BV2, that specifically rec-ognize cells undergoing developmental programmed cell deathin different tissues of the chicken and zebra-finch embryos. Theantigen recognized by BV2 monolonal antibody is detected invitro in primary chicken embryonic fibroblasts induced to die byactinomycin D, as well as fibroblasts induced to die by chemicalanoxia. The expression of this specific antigen during necrosisappears to require active protein synthesis. These dinpsprovide evidence that cells from different embryonic tissuesundergoing prgrammed cell death during vertebrate develop-ment express simlr antigens and indicate that apoptosis andnecrosis may share similar biochemical features.

The morphological concept of apoptosis, defined as a phys-iological type of cell death, has progressively been used as asynonym with the term programmed cell death, originallydescribed during normal development (1) and implying theexistence of a genetic program of cell death (2). Apoptosis isbelieved to account for most cell death during developmentand in normal adult tissue turnover, and it can also be inducedexperimentally by various biological, chemical, or physicalagents (3). Necrosis, in contrast, has been defined as apassive degenerative phenomenon and is observed in a tissuesubjected to direct toxic or physical injury-such as hyper-thermia, hypoxia, and ischemia or to complement-mediatedlysis (4-7). To investigate the mechanism of cell death, wehave identified a monoclonal antibody (mAb), BV2, thatspecifically recognizes cells undergoing programmed celldeath in developing chicken embryos, and we used BV2 mAbas a specific marker of chicken programmed cell death tostudy the relationship between apoptosis and necrosis.

MATERIALS AND METHODSGeneration of mAbs. Chicken eggs were purchased from

Spafas, Norwich, CT. Zebra finches (Poephila guttata) weregrown in the avian facilities of the California Institute ofTechnology. Chicken embryos were staged according to V.Hamburger and H. L. Hamilton (8). The generation of thecell-death-specific mAbs by neonatal tolerization (9) is de-scribed in detail elsewhere (10). BV2 hybridoma was screenedon sections of day 7 chicken hind limb foot plates, expanded,and assayed several times for antibody reactivity and stabi-lized by three cycles of cloning by limiting dilution. Theimmunoglobulin (IgM) type was determined by ELISA usingrat mAb anti-mouse immunoglobulin classes from Amersham.BV2 hybridoma cells (2-5 x 106) were injected into 7- to

8-week-old F1 BALB/cJ x C57/BL male mice (The JacksonLaboratory) primed with incomplete Freund's adjuvant (Cap-pel), and 2 weeks later, ascites fluid was collected and purifiedby E-Z-Sep (Middlesex Sciences, Foxborough, MA).

Immunofluorescence. Whole embryos and limbs wererinsed in cold phosphate-buffered saline (PBS) and fixed in4% periodate/lysine/paraformaldehyde prepared in PBS for12 hr at 4TC. Tissues were then washed in cold PBS anddehydrated in graded concentrations ofethanol (50%-95%) at4TC. Infiltration was conducted with solutionA ofJB-4 glycolmethacrylate embedding kit (Polyscience) in a light-protectedglass vial at 40C for several days (11). Tissues were embeddedin glycol methacrylate and placed under vacuum (15 mmHg;1 mmHg = 133 Pa) at 4°C and allowed to polymerize for 48hr at 4°C (12). Sections were cut at 5 ,urm with glass knives ona Sorvall JB-4 microtome (DuPont), transferred via water toSuperfrost/plus pretreated slides (Fisher Scientific), andair-dried at room temperature. For immunofluorescencestaining, the sections were treated with xylene for 30 min atroom temperature, extensively washed with cold PBS, anddigested with Pronase at 0.5 mg/ml (Sigma)/PBS for 1 hr at3rC. The enzyme reaction was stopped with cold 0.5 M Trisbuffer on ice followed by washing in cold PBS. The slideswere then treated with a blocking solution consisting of 1%bovine serum albumin (Sigma) and 1% heat-inactivated nor-mal goat serum (GIBCO) in PBS for 30 min at room temper-ature. Incubation was conducted for 5 hr at room temperaturewith the BV2 ascites fluid diluted in blocking buffer. Afterseveral washes with PBS, affinity-purified goat anti-mouseantibody conjugated with fluorescein (1:100, Cappel) wasdiluted into blocking buffer and applied to the slides for 1 hrat room temperature together with Hoechst 33258 (Ho 33258)dye (0.5 jug/ml, Sigma), a DNA minor groove-binding ligandthat has strong adenine plus thymidine sequence specificity(13). After washing with PBS, the slides were mounted in 10oPBS/90% glycerol with p-phenylenediamine at 1 mg/ml(Sigma) to prevent photo bleaching. To verify the specificityof the antibody labeling, control staining was done withpreimmune or nonimmune immunoglobulins as primary an-tibodies or with secondary antibody alone. No significantstaining was obtained with these controls, even in compacttissues. Positive controls included several antibodies specificfor different chicken structures. Serial sections were coun-terstained by methylene blue-azure II and basic fuchsin (14).Sections were examined with a Zeiss epifluorescence Axio-plan microscope with x20, x40, and xlO0 objectives and afilter set and photographed by using Kodak 1600 ASA film.

Cell Cultures. Primary chicken embryo fibroblast cultureswere freshly prepared for every experiment from 9-day-oldembryos (15). Cells were plated out at different densities onculture slides with four chambers (Nunc) coated with type 1

Abbreviation: mAb, monoclonal antibody.§To whom reprint requests should be addressed.

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8642 Cell Biology: Fernandez et al.

collagen (Sigma, 500 Al of a 50 ,ug/ml solution for 8 hr) andwere cultured in F-12 medium (GIBCO)/10% fetal bovineserum (HyClone)/2.5% chicken serum (Sigma), penicillin at100 units/ml (GIBCO)/streptomycin at 100 ,ug/ml (GIBCO)at 370C in an atmosphere of5% C02/95% air in a humidifiedincubator. To mimic a state of cell anoxia, 1 mM KCN and2 mM iodoacetate were added to the culture medium. Celldeath was also induced with 10-6 M actinomycin D, andinhibition of protein synthesis was done with cycloheximideat 50 Ag/mI (all chemicals from Sigma). Loss of cell viabilitywas determined by failure to exclude Trypan blue and bymeasuring the leakage of L-lactate dehydrogenase (EC1.1.1.27) by Sigma procedure no. 500. For antibody staining,cell cultures were rinsed in cold PBS, fixed in freshlyprepared 4% periodate/lysine/paraformaldehyde in PBS for15 min at room temperature, and then rinsed twice in coldPBS for 5 min. Cells were permeabilized by treatment with0.2% Triton X-100 in PBS at room temperature for 2 min (topreserve the cytoskeleton structure) to allow penetration ofantibodies into the cells and eliminate the danger of furtherosmotic damage. Cell culture slides were then rinsed withPBS, and immunofluorescence staining was done as de-scribed. Actin filaments were stained with tetramethyl-rhodamine B isothiocyanate-phalloidin (10-7 M, Sigma).Rhodamine-conjugated wheat germ agglutinin (10 jug/ml,Sigma) was used as a marker for the Golgi apparatus becauseof its high affinity for the terminal glucosamine residuespresent in incompletely glycosylated proteins (16).DNA Fragmentation Assay. After treating primary chicken

embryonic fibroblasts for 24 hr (1 x 107 cells per 10-cm dish)with actinomycin D (2.5 x 10-7 M final concentration),

adhering cells were harvested by trypsinization and com-bined with floating cells present in the supernatant. Aftercentrifugation, the cell pellet was lysed in extraction buffer(100mM NaCl/25 mM EDTA/10mM Tris HCl, pH 8.0/0.5%SDS/proteinase K at 100 pg/ml for 3 hr at 55°C, extractedtwice with phenol/chloroform and once with chloroform; theDNA was then ethanol-precipitated overnight at -80°C. Theprecipitated DNA was dissolved in 10 mM Tris, pH 8/1 mMEDTA containing RNase A at 10 pg/ml and analyzed on a1.7% agarose gel. Electrophoresis was done for 3 hr at 140 V,and the DNA was visualized by staining with ethidiumbromide. DNA from untreated chicken embryonic fibroblastswas extracted in parallel and served as a control.

RESULTSIsolation of the mAb. The mAb BV2 was isolated, after

neonatal tolerization, by immunization of mice with embry-onic day 7 (stage 31) chicken foot plates. At this stage,developmental programmed cell death is easily identifiable inthe interdigital mesenchymal areas of foot plates (Fig. 1A).Interdigital programmed cell death, a major process duringthe morphogenesis of digits in all nonwebbed amniotes,including the human, plays an important role in the separa-tion ofthe chondrifying digits (17, 18). To selectively enhancethe production of antibodies specific for dying cells, themouse immune response to living-cell antigens was sup-pressed by injecting newborn mice with day 10 chicken footplate tissues (with virtually no cell death) before immuniza-tion. Potential cell-death-specific antibodies were screenedby using indirect immunofluorescence staining on sections of

FIG. 1. Immunofluorescence staining of BV2 mAb on 5-,um glycol methacrylate sections of the chicken embryonic hind limb foot plates.(A) Structure of the hind limb foot plate at stage 31-32 (day 7) stained with methylene blue-azure II and basic fuchsin. Mesenchymal apoptoticdying cells are stained dark blue in the interdigital areas. (B-I) Double-staining immunofluorescence pictures: (B, D, F, H) Ho 33258 stainingofthe nuclear chromatin; (C, E, G, I) BV2 mAb staining ofcorresponding Ho 33258 dye staining ofB, D, F, and H, respectively. (B) Enlargementof the interdigital area of the hind limb foot plate shown in A. Nuclei of dying cells appear as shining condensed and fragmented dots and arenot present in the digit (d). (C) BV2 mAb staining on the same section as in B. (D and E) Detection ofBV2 mAb staining along the distal edgeof the apical ectodermal ridge of the limb bud at stage 23-25 (day 4). (F and G) BV2 staining of the anterior necrotic zone of the chicken embryohind limb at stage 23-25 (day 4). (H and I) Enlargement of the death area shown in F and G with apoptotic bodies with condensed shiningchromatin (H) and the corresponding staining with BV2 mAb (I). Some degenerating cells lie among normal cells, but the majority are aggregatedin the cytoplasm ofan engulfing cell (2, 17). Some ofthe dead cells in Ho 33258 dye staining and BV2 mAb staining are marked with small arrows.BV2 mAb staining is not detected in living cells with normal nucleus; nor is it detected in mitotic figures (m). [Bar = 445 pm (A), 220 Pm (Band C), 56 ,um (D-G), and 26 am (H and I).]

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day 7 chicken foot plates. Cells undergoing programmed celldeath show typical nuclear condensation and fragmentation,which can be revealed by costaining with Ho 33258 dye (Fig.1B and H). BV2 mAb was one of several mAbs isolated in thisway that showed specific staining of dying cells with apo-ptotic morphology (Fig. 1 C and I).

Speciflcity of the Antibody in Vivo. To determine thespecificity of BV2 mAb, we stained glycol methacrylatesections ofday 3 and 4 chicken embryo and ofday 15 chickenretina with BV2 mAb and Ho 33258 dye by using immuno-fluorescence. The staining of BV2 mAb in the developingembryo was evident in many different areas of cell deathpreviously mapped by embryologists by vital staining in ovo(17,18). In the hind limb bud, BV2 mAb staining was detectednot only in the mesenchymal interdigital areas at stages 31-32(day 7) (Fig. 1 B and C) but also at stages 23-25 (day 4) in theanterior necrotic zone (Fig. 1 F-I) in the centrally locatedopaque patch (Fig. 2 A and B) and in the posterior necroticzone (data not shown). These mesodermal areas of cell deathare probably involved in the regulation of the limb size bycontrolling the amount of distal mesenchyme available fordigit formation and in skeletal patterning, such as the initialseparation of tibia and fibula (18). Another area of cell deathin the limb that was stained with the antibody was along theedge of the apical ectodermal ridge (Fig. 1 D and E), athickening of the ectoderm of the distal end of the limb budthat is known to induce the outgrowth of the underlying

mesoderm (17). Similar staining could be detected in manystructures derived from both mesodermal and endodermalorigin-for example, in the tail bud (Fig. 2 A and B), themesonephros and collecting ducts (Fig. 2 C and D), the gut(Fig. 2 G-I), the mesenchyme of the mandible, the wing bud,around vessels, and in the heart (data not shown). Cell deathwas intense in the mesenchymal regions undergoing activeproliferation and migration, such as the sclerotomes of day 4embryos (Fig. 2 A and B, as well as J-L). In all these areas,the cellular fragments of dying cells stained by the antibody,were generally aggregated (Fig. 1 H and I), most likely in thecytoplasm of an engulfing cell (2, 17). Cell death is also amajor morphogenetic feature in neurogenesis (19). BV2 an-tigen could be detected in the neuroectoderm, in cells withpyknotic nuclear morphology revealed by Ho 33258 staining.Fig. 2 (E and F) shows the staining in the bipolar layer of theembryonic retina at day 4. BV2 mAb was then tested onfrozen sections of different avian species and showed intensecross-reactivity in zebra-finch embryos and similar patternsofexpression in different embryonic tissues (data not shown).All areas of pyknosis detected in the embryos with Ho 33258dye on serial sections of 5 ,m were stained with the mAb,without exception. No staining was detected in living cells orin mitotic figures (Fig. 1 H and I). Taken together, theseresults indicate that cells undergoing programmed cell deathin avian embryonic tissue, derived from the three embryonicgerm layers, actively express at least one similar antigen.# ^, . .. ,b,, _a . . I . ..^ .S., .t _, tt, n

r ^ A, SV ^ aS__ _ .w

FIG. 2. Detection of BV2 mAb staining in different tissues of the chicken embryo derived from all three embryonic germ layers. Picturesare from 5-,um glycol methacrylate parasaggital sections of stage 23 to 25 (day 4) embryos, stained with methylene blue-azure II and basic fuchsin(G, J) or by double immunofluorescence staining with chromatin dye Ho 33258 (blue) and BV2 mAb (green). (A and B) General view of theposterior part of a chicken embryo showing the extension of cell death during embryonic development. All areas of pyknosis detected by thedye are stained with the mAb. Cell death is intense in the somitic derived mesoderm (s) and in the tail bud (t). An important area of cell deathat the base of the primitive limb bud (I) is probably related to the centrally located opaque patch (k, mesonephros). (C and D) Double stainingof intensive cell death with BV2 mAb and Ho 33258 in mesonephric ducts (d) (t, mesonephric tubules). (E and F) Double staining of BV2 mAband Ho 33258 of a section of neuroectoderm: BV2 staining in embryonic retina (r), at the level of the optic nerve (on). In the nervous tissue,the pyknotic bodies stained with the dye or the mAb are more dispersed than in the mesodermal-derived tissues (see for example, Fig. 1 F andG). (G-I) Histological staining (G), Ho 33258 (H), and immunofluorescence of BV2 mAb (I) of a section of endoderm: dying cells in theepithelium of the hind gut endoderm (g) are evident. (n, Neural tube; nt, notochord; v, blood vessels). (J-L) Histological staining (J), Ho 33258staining (K), and immunofluorescence of BV2 mAb (L) of cell-death areas in the sclerotome (s). (dm, Dermomyotome; 1, limb; dorsal is up.)[Bar = 220 Am (A, B, G, and J), 130 ,um (E and F), and 110 ,um (C, D, H, I, K, and L).]

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Detection of BV2 mAb Durng Celi Death in Vitro. To studypossible conditions that specifically induce the expression ofthe antigen recognized by BV2 mAb, we analyzed chickenembryonic fibroblasts undergoing cell death in vitro. Nuclearand cytoplasmic morphological changes were followed bystaining the chromatin with Ho 33258 dye and staining theactin filaments with phalloidin. In several situations in vivo orin vitro, apoptosis can be suppressed or postponed by inhib-itors ofRNA or protein synthesis, suggesting that cell deathis an active process requiring new gene expression. In otherexamples, however, cells undergo apoptosis in the presenceof protein or RNA synthesis inhibitors (20). In the latterinstances, presumably, proteins that suppress cell death needto be constantly synthesized while cell-death machinery isalready in place. We induced cell death ofchicken embryonicfibroblasts in vitro by addition of actinomycin D, an inhibitorofRNA polymerase and a cancer chemotherapeutic agent (3).Nuclear and cytoplasmic condensation of the cells in vitrocan be observed 2 to 3 hr after treatment (Fig. 3 G and H).Genomic DNA isolated from such cells showed typical DNAfragmentation patterns (data not shown). BV2 mAb stainingwas detected in dying cells 6-8 hr after drug addition (Fig.31). No staining was detected in control living fibroblasts(Fig. 3C). Thus, the antigen recognized by BV2 mAb isexpressed in dying cells in vivo and in vitro.

Detection of BV2 mAb During Necrosis in Vitro. The othercell death condition we tested was chemical anoxia inducedby potassium cyanide and iodoacetate, which serve as typicalexamples of necrotic stimuli (21-23). Upon addition of thesemetabolic inhibitors of respiration and glycolysis, chickenfibroblasts grown on collagen swell and detach in <3 hrwithout nuclear condensation (Fig. 3 D and E), as describedfor necrosis (2, 21). The antigen recognized by the BV2 mAbwas dramatically induced in dying fibroblasts =20 min afterthe induction of necrosis (Fig. 3 F and K). Removal ofcyanide and iodoacetate after 20 min did not rescue the dyingcells, nor did it suppress expression ofthe antigen recognizedby BV2 mAb. BV2 staining of necrotic cells was in certainlocalized regions of cytoplasm, in contrast to the staining incell death induced by actinomycin D. Rhodamine-conjugatedwheat germ aggiutinin was used as a marker for the Golgiapparatus because of its high affinity for the terminal glucos-amine residues present in incompletely glycosylated proteins(16). Analysis of the BV2 mAb staining in necrotic cells, bycostaining with the lectin, suggested that the antigen recog-nized by BV2 mAb may be localized in the Golgi apparatus(Fig. 3 J-L). Pretreatment of the cultures with cycloheximide(50 ptg/ml for 2 hr), an inhibitor of protein synthesis, com-pletely suppressed BV2 staining, even though cells stilldetach and die as untreated necrotic fibroblasts (data notshown). The brief treatment of cells with cycloheximide did

FIG. 3. Correlation between cell death in vitro induced by actinomycin D and necrosis using BV2 mAb staining. All views are tripleimmunofluorescence staining pictures of chicken embryonic fibroblasts grown on collagen and induced to die by drug treatment. Ho 33258staining of the chromatin is shown in blue; BV2 mAb staining is shown in green. Phalloidin staining of actin filaments (B, E, and H) or wheatgerm agglutinin staining of the Golgi apparatus (L) is shown in red. (A-C) Control cells. BV2 staining (C) was not detectable on untreatedfibroblasts. (D-F) Necrosis. Fibroblasts were induced to die by chemical anoxia after exposure to 1 mM potassium cyanide and 2 mMiodoacetate. BV2 mAb staining is detected 20 min after induction of necrosis (F). (G-I) Fibroblasts were induced to die by exposure to 10-6M actinomycin D. Nuclear (G) and cytoplasmic (H) condensation are seen 2-3 hr after drug addition. (I) BV2 mAb staining, detected after 6-8hr. (J-L) Localization of BV2 antigen in the Golgi apparatus (K) during necrosis by costaining with wheat germ lectin (L). [Bar = 70 pm (A-Cand G-I), 56 ,m (D-F), and 28 pLm (J-L).]

Proc. Natl. Acad. Sci. USA 91 (1994)

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not affect the viability of the control chicken fibroblasts.Thus, the expression of the antigen recognized by BV2 mAbin cells undergoing necrosis appears to require protein syn-thesis, rather than unmasking of preexisting determinant,although the determinant itself could be a carbohydrate orany other protein modification.

DISCUSSIONThe molecular mechanisms of cell death in vertebrates arestill largely not understood. In the nematode Caenorhabditiselegans, a genetic pathway of programmed cell death hasbeen described. Eleven genes identified in this pathway affectall cells that undergo programmed cell death during C.elegans development (1), suggesting that all cells undergoingprogrammed cell death share a common mechanism. Manycells die at predicted times during vertebrate development,but it is not clear how many different mechanisms haveevolved to execute cell death in vertebrates. Expression ofthe antigen recognized by BV2 mAb is specifically detectedon apoptotic cells in all the areas of cell death in developingchicken embryos and zebra-finch embryos so far examinedby us, suggesting that during vertebrate development, pro-grammed cell death in tissues derived from all embryonicgerm layers may share a common mechanism as well. Ex-pression of several genes has been associated with experi-mentally induced apoptosis or developmental cell death, butthe expression of all those genes have been detected in livingcells as well (24-31). In contrast, based on our developmentalstudies, using immunofluorescence techniques, BV2 mAbappears as a different specific marker of apoptotic cellsduring avian development. Although it is unlikely that theantigen recognized by BV2 mAb is directly involved incontrolling cell death, because the inhibition of its expressionduring necrosis by cycloheximide does not block cell death,the identification of a specific marker of cell death will allowus to characterize cell death under different conditions.The unexpected result that the expression of the antigen

recognized by BV2 mAb is also induced during cell deathcaused by chemical anoxia and its dependency upon proteinsynthesis suggests that these types of necrosis and apoptosismay share certain common biochemical elements. The endog-enous machinery of apoptosis may also be activated andcontribute to cell death under certain necrotic conditions.Overexpression of the bcl-2 gene has been found to not onlyinhibit cell death induced by serum or growth factor depriva-tion but also protect certain vertebrate cells from sodiumazide, glucocorticoids, phorbol ester, methotrexate, heatshock, ethanol, radiation, antimetabolite 1-#-D-arabinofura-nosylcytosine, calcium ionophore, vincristine, glucose with-drawal, membrane peroxidation, free radical-induced damage,and tumor necrosis factor-mediated cytotoxicity (32-39).Thus, some of the apparent differences between apoptosis andnecrosis on the basis of morphological criteria may not bereflected entirely on a mechanistic level, and both pathwaysmay share certain common molecular players. If so, ourfindings suggest that one should look at traditional necrotic celldeath, such as cell death during ischemic diseases ofthe heart,kidney, and brain, the primary causes of mortality and mor-bidity in industrialized nations (40), in a different way.

P.-A.F. and R.R. contributed equally to this paper. We are gratefulto S. Hockflield for many valuable insights on neonatal tolerization andgeneration of mAbs, to E. Harlow for suggestion on generation ofmAbs, and to H. R. Horvitz for helpful comments during manuscriptpreparation. We thank V. Gagliardini, C. Tuffereau, C. Fankhauser,M. Igarashi, M. Miura, M. Pack, and C. Simpson for stimulatingdiscussions. J.Y. was supported by a grant from the National Instituteof Aging (AG11017) and a grant from Bristol-Myers/Squibb. P.-A.F.was supported by a fellowship from Rh6ne-Poulenc.

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