0892-6638/97/001 1-0801/$02.25 C FASEB 801

Apoptosis in Xenopus tadpole tail muscles involves Bax-dependent pathways

LAURENT M. SACHS,* BASSIMA ABDALLAH,* AHMED HASSAN,* GIOVANNI LEVI,AMAURY DE LUZE,* JOHN C. REED,t AND BARBARA A. DEMENEIX*.’

*Laboratoire de Physiologie G#{233}n#{233}raleCt Compar#{233}e,Museum National d’Histoire Naturelle, URA

CNRS 90, 75231 Paris, cedex 5, France; tLabtratoiy of Molecular Biology, CBA-IST, Genoa, Italy; andtThe Burnham Institute, La Jolla, California 92037 USA

ABSTRACT Apoptosis is a fundamental media-nism implicated in normal development. One of themost spectacular developmental events involvingapoptosis is tail regression during amphibian meta-morphosis. We analyzed how thyroid hormone (3,5,3’-triiodothyronine, ‘F3), the orchestrator of meta-morphosis, affects expression and function of theproapoptotic gene Bax in the tail muscle of free-livingXenopus tadpoles. During natural metamorphosis BaxmRNA was expressed in tail muscles and was spatiallycorrelated with apoptosis. Precocious treatment oftadpoles with T3 induced Bax expression and apopto-sis. To verify that Bax expression was causally relatedto apoptosis we used a naked DNA gene transfermethod to express Bax in the dorsal tail muscle. Thisinduced apoptosis, and the process was exacerbatedby T3 treatment. To determine whether T3 effects onBax expression involved transcriptional regulation,we injected a Bax promoter sequence into dorsal andcaudal tail muscles. In the dorsal muscle, ‘F3 treat-ment did not affect transcription from the Bax pro-moter. However, in the caudal muscle, T3 treatmentsignificantly increased Bax transcription. We con-clude that Trinduced apoptosis in Xenopus tadpoletail muscles involves Bax.activating and Bax-synergis-

tic mechanisms. These programs are induced in spa-tially and temporally distinct manners.-Sachs, L M.,Abdailah, B., Hassan, A., Levi, G., de Luze, A., Reed,J. C., Demeneix, B. A. Apoptosis in Xenopus tadpoletail muscles involves Bax-dependent pathways. FASEBJ. 11, 801-808 (1997)

Key Words: thyroid honnone metamorphosis in vivo genetransfer

ApOPTOSIS IS A HIGHLY CONSERVED, genetically gov-erned response for cells to commit suicide (for a re-cent review, see ref 1). Regulation of cell death is ascritical to development as regulation of cell prolif-eration; in the mature animal, maintenance of ho-meostasis implies balancing cell production and cell

elimination (2). Deregulation of apoptosis can leadto impaired development and pathologies such as tu-morigenesis.

One of the first systems in which apoptosis was de-scribed was the regressing tail of metamorphosingtadpoles (3). Indeed, amphibian metamorphosis,which is orchestrated by thyroid hormone (3, 5, 3’-triiodothyronine or T3) ,2 is one of the best-studiedhormone-regulated developmental processes (4, 5),and it provides an excellent experimental model forstudying apoptosis (6). First, the free-living animal

can be easily manipulated; second, the process canbe induced (or blocked) by modif’ing the thyroidstatus of the tadpoles. Moreover, in vivo gene transfercan be applied to the tail muscle tissue to study generegulation and function (7, 8).

Apoptosis proceeds in three stages: triggering,judgment, and execution. Numerous genes play keyroles at each stage. Triggers vary widely according tocell type and developmental stage. In the judgmentor decision phase, the Bcl-2 family of genes is seen asproviding central checkpoints (9). Last, the cysteineproteases or caspases are key players in execution.Our interest focused on the checkpoint process andgenes related to the proto-oncogene Bc12. Proteinsencoded by these genes can act as repressors (Bcl-2,Bcl-xL, Mci-i) or effectors (Bax, Bak, Bad, Bcl-xs) ofapoptosis (10). Each protein has a highly conserveddimerization domain that enables the proteins to in-teract by forming homodimers or heterodimers, orboth. Current thinking holds that, since in most ex-perimental situations Bcl-2 protects cells and Bax in-

duces apoptosis, then Bcl-2 and other anti-apoptoticmembers of the family compete for the binding ofBax in order to inactivate it (10).

Bax has been described as an effector of apoptosisin many cellular systems (ii). In keeping with thishypothesis, Bax null mice display lymphoid hyperpla-sia (12). However, little in vivo data are availableabout the involvement of the protein in apoptosis in

‘Correspondence: Laboratoire de Physiologie G#{233}n#{233}raleetCompar#{233}e,Museum National d’Histoire Naturelle, URACNRS 90, 7, rue Cuvier, 75231 Paris, cedex 5, France

Abbreviations: T,, 3,5, 3’-triiodothyronine; GFP, green flu-orescent protein; ISH, in situ hybridization; p.i., postinjection;ECM, extracellular matrix; TR, T5 receptor; TUNEL, terminal

deoxytransferase-mediated dUTP-biotin nickend labeling.

802 Vol.11 August 1997 The FASEB journal SACHS ET AL.

other vertebrate systems. We chose to follow endog-enous Bax expression and then examine the conse-quences of expressing Bax in the tail muscle ofpremetamorphic tadpoles. We found that it did in-duce apoptosis and that the apoptotic effect was ex-acerbated by T3. We conclude that 1) Bax expressionis induced in the tail during metamorphosis, 2) thisinduction is correlated with apoptosis, and 3) T3 in-duces apoptosis by activating Bax-dependent path-ways.

MATERIALS AND METHODS

Anhnak

Xenopus laevis tadpoles were raised and maintained as previ-ously described (7) and staged according to Nieuwkoop andFaber (13). At stages 50-51, before prometamorphosis, theywere transferred to 0.1% sodium perchlorate (Carlo Erbareactifs, Nanterre, France) to block development. For in situhybridization and TUNEL (terminal deoxytransferase-medi-ated dUTP-biotin nickend labeling), we used albino tadpolesobtained from the Service d’Elevage de X#{233}nopedu CentreNational de Ia Recherche Scientifique (Rennes, France).Metamorphosis was induced by adding 10 nM T, (Sigma, St.Quentin Fallavier, France) to the aquarium water that waschanged daily.

Plasmids and in situ hydridization (ISH) probes

Plasmids were propagated and purified by standard CsC1 tech-niques (14). pcDNA3-LUG (kindly provided by Dr. M. Schleef,Quiagen), the firefly luciferase activity, is driven by the cyto-megalovirus (CMV) promoter of pcDNA3 (Invitrogen, San Di-ego, Calif.). PcDNA3-Bax was constructed by subcloning theEcoRI-EcoRI fragment of mouse Bax cDNA in the EcoRI siteof pcDNAS. The mouse Bax cDNA (70Z7) (15) was a gift fromDr. S. J. Korsmeyer (Howard Hughes Medical Institute, St.Louis, Mo.). PcDNA3-GFP was constructed by subcloning the

EcoRI-XhoI fragment of pRSET B-S65T containing the S65Tgreen fluorescent protein (GFP) mutant cDNA (16) in theEcoRl-XhoI sites of pcDNAS. The GFP mutant clone was pro-

vided by Dr. R. Y. Tsien (Howard Hughes Medical Institute,La Jolla, Calif.). pcDNA3-xp53 was obtained by subcloning theEcoRl-EcoRI fragment of Xenopus p53 B2 clone (17) into theEcoRI site of pcDNA3. The pBluescript-xp53 clone was a giftfrom Dr. M. Mechali (InstitutJacques Monod, Paris, France).pSV4OxTF43: The full-length cDNA of Xenapus TRI3 was clonedinto the pTL1 expression vector (18). This plasmid was kindlyprovided by Dr. J. R. Tata (Medical Research Council, Lon-don, U.K.). pTM667-3: The 371 bp fragment of the humanbax promoter between -318 bp and -687 bp was subclonedinto the Hindlil site of pUCSVOCAT (19). pBluescript-Bar.Thefirst 502 bp of the fragment EcoRI-BamHI of mouse BaxcDNAwas subcloned into the EcoRI-BamHI site of pBluescript(SK+). This construction provides an antisense probe with T3RNA polymera.se and a sense probe with T7 RNA polymerase.

In vivo gene transfer

Somatic gene transfer in Xenapus tadpole dorsal muscle wasperformed as described previously (7). The same methodol-ogy was used for in vivo gene transfer in the caudal muscle.The injection solution (1 p3) contained various amounts of

pure DNA (between 1.1 and 2.6 jig) in 0.07 M NaCI colored

with fast green (Sigma, St Quentin Fallavier, France). For stud-ies involving quantification of promoter activity, transfection

efficiency was normalized using a constituitive construct,pcDNA3-LUC.

Reporter gene assays

Luciferase and CAT assays were carried out according to deLuze et al. (7). In order to follow GFP expression, the livetadpole, anesthetized in MS222 (0.1%, Sandoz, Basel, Switzer-land), was placed directly under an Olympus fluorescent mi-croscope illuminated with a 395 nm ultraviolet excitationbeam, and emission was observed at 509 nm.

In situ hybridization

In situ hybridization (ISH) probes were synthetized using T3(for Bax antisense probe) and T7 (for Bax sense probe) poly-merase (Boehringer, Mannheim, Germany), as previously de-scribed (8). Before the last step of purification by ammoniumacetate, the probe was fragmented by alkaline lysis in bicar-bonate buffer (NaHCO, 0.2 M, Na2CO, 0.2 M, pH 10). Lo-calization of Bax mRNA transcripts was established onlongitudinal cryostat sections by in situ hybridization, as pre-viously described by Whitfield and co-workers (20). Slideswere dipped in NTB2 photographic emulsion (Kodak), leftfor 2 wk, and then developed. After dehydration, slides werestained with hematoxylin/eosin, photographed, and the graindensity quantified by computerized videomicroscopy using adigital image processor (Optilab program from Graftek run-ning on a Macintosh Power computer). The selected micro-scopic image was digitalized under optimal conditions ofillumination and contrast. All values were corrected for back-ground with sense probe.

TIJNEL technique

The TUNEL procedure was performed on sections sequentialto those used for ISH. A commercial in situ cell death detec-tion kit (1 684 809, Boehnnger-Mannheim) was used. Afterlabeling, the slides were dehydrated, stained with hematoxy-un, and mounted. Quantification of cell death was carried outby counting the red (alkaline hosphatase positive) stainednuclei and normalized per mm.

unmunoprecipitation and Western blotting

We used rabbit anti-peptide antisera specific for amino acids43 to 61 of the mouse Bax protein (21). For immunoprecipi-tation, 10 dorsal muscles cotransfected with 1 jig of pcDNA3-LUG and 1 jig of pcDNA3-Bax were sonicated on ice in 1 mllysis buffer (10 mM Tris, pH7.5,.150 mM NaCl, 5 mM EDTA,1% Triton X100, 1 mM phenylmethylsulfonyl fluoride, 5 jig/ml Aprotinin, 2 jig/mi Leupeptin and I jig/mi Pepstanin).Lysates were treated according to Miyashita and collaborators

(22) before PAGE. After transferral to a prewet PVDFmembrane (BioRad) and incubation (overnight, 4#{176}C)inblocking buffer (Tris HCI 50 mM, pH 7.6, NaCl 200 mM, gel-atine 0.25%), it was incubated with anti-bax (1/3000, over-night, 4#{176}C).Antibodies were detected using an ECL kit fromAmersham (Arlington Heights, Ill.).

E

15

54 Dev. Stage (NF) 61 63 65

T3, BAX, AND APOPTOSIS IN XENOPUS TADPOLES 803

RESULTS

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Endogenous Bax expression is induced duringnatural metamorphosis

We followed endogenous Xenopus Bax expressionand its eventual correlation with apoptosis duringmetamorphosis. Given the high identities betweenBax genes of different species (89.4% betweenmouse and human), we carried out ISH with a

probe derived from the mouse Bax gene. The speci-ficity of the signal obtained was verified with a senseprobe (see Materials and Methods). Apoptosis wasfollowed by the TUNEL technique (23). Controlsincluded eliminating the terminal transferase orUTP-biotin, or both. ISH probes for Bax mRNA and

Figure 1. Bax expression andapoptosis are spatially corre-lated in caudal muscle duringnatural metamorphosis. En-

dogenous Bax mRNA expres-sion (A, B) was followed byISH, using an antisense 32P-la-beled mouse Bax probe. Apop-tosis (C, D) was followed by aTUNEL technique with alka-line phosphatase-linked strep-tavidin to reveal incorporatedUTP-biotin. A, C) Sequentialsections of the caudal muscle

from a tadpole in premetamor-phosis at st54 showing no hy-

bridization signal (A) and nostained apoptotic nuclei (C). B,D) Sequential sections of a typ-ical area of caudal muscle froma tadpole at metamorphic cli-max (st6l) showing a denseBax signal (B) and numerousapoptotic nuclei labeled red byalkaline phosphatase (D, short,thick arrows). X3000. Thin ar-rows indicate normal nucleistained with hematoxylin. E)Quantification of ISH (opencolumns) and TUNEL signal(filled columns) in sections ofcaudal muscle from tadpoles at

different stages of metamor-phosis. The drawing representsa st54 tadpole and the rectan-gle indicates the area quanti-fied. The levels of endogenousT3 indicated are adapted fromLeloup and Buscaglia (24).Means ± SEM are given; n a 6in all cases (three sections oftwo tadpoles).

TUNEL were carried out on sequential sections oftails from tadpoles in premetamorphosis (stage 54,or st54), metamorphic climax (st6l), and duringmaximal tail regression (st63). st6l corresponds tothe T3 peak during metamorphosis (Fig. 1E, datafrom Leloup and Buscaglia (24)). Endogenous Xeno-

pus Bax mRNA and numbers of apoptotic nuclei were

lower during premetamorphosis (Fig. 1A, C) than atst6l (Fig. lB. D) or st63 (Fig. 1E). The greatest in-

crease in numbers of apoptotic nuclei per mm2 oc-curred between stages 61 and 63 (P<0.0l, Fig. 1L),

corresponding to the maximal rate of tail regression.Quantif’ing apopto tic nuclei within different parts of

the tail showed that apoptosis progressed in a caudalto rostral direction with advancing developmental

stage (data not shown).

I

pcDNA3-LUC) into dorsal muscle. Final activity of thereporter provides an index of cell survival (25). WhenGFP was used as the indicator of cell survival in free-living animals, we found continued GFP expressionover several months in muscle fibers of all tadpoles

injected with GFP plus a control plasmid (Fig. 3A)and no GFP expression in any animal injected withGFP plus a Bax vector. This naked DNA transfermethod results in transgene expression in numerous

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Figure 2. T3 treatment induces endogenous Bax mRNA andapoptosis in the caudal part of the tail, but not the dorsalmuscle. A) Densitometry was used to quantify the Bax mRNAISH signal in different regions of cryostat sections of tail mus-cle from control tadpoles and animals that had received T3treatment for 48 h. B) The number of apoptotic nuclei perunit area was measured in the same regions ofTUNEL-labeledsections from control and T3-treated (48 h) tadpoles. Thedrawing represents a st54 tadpole in which the areas quanti-fied are indicated. Means ± SEM are given; n�6 in all cases.

T3 increases endogenous Bax expression in caudalbut not dorsal muscle

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24h 48h 72hTime post-injection

804 Vol. 11 August 1997 The FASEB journal SACHS ET AL.

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T3 effects on endogenous Bax mRNA expression andthe apoptotic process were examined in dorsal andcaudal tail muscles using tadpoles blocked in pro-metamorphosis by anti-thyroid treatment, therebyavoiding interference from endogenous T3. After T3treatment, Bax mRNA (Fig. 2A) and apoptotic nuclei(Fig. 2B) increased more than sixfold in the caudalmuscle but not in the dorsal muscle.

Overexpressing Bax induces apoptosis in dorsal tailmuscle

To determine Bax effects in tail muscle, we coin-jected an expression vector encoding murine Bax

and a constituitive reporter vector (pcDNA3-, GFP, or

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F’Figure 3. Bax transfection reduces expression of cotransfectedGFP or Iuciferase in dorsal muscle in vivo. A) Detection of thekilling activity of Bax by GFP cotransfection. Tadpoles werecotransfected in the dorsal muscle with I jig GFP marker plas-mid and either 1 jig of a control plasmid vector or a plasmid-expressing mouse Bax. Animals were photographed under anOlympus fluorescent microscope with a lOX objective usingexcitation/emission filters as for fluorescein. Because only su-perficial fibers could be focused correctly, some of the otherdeeper situated GFP-positive fibers are indicated by white ar-rows. Usually up to 15 GFP-positive fibers were found at 72 hpostinjection. No GFP signal was see in tadpoles injected withBax. B) Detection of the killing activity of Bax by luciferasecotransfection. Tadpoles were cotransfected in the dorsal mus-

cle with 1 jig luciferase marker plasmid and either I jig of acontrol plasmid (vector) or a plasmid expressing mouse Bax.

Animals were killed 24, 48, or 72 h later and luciferase wasassayed in muscle homogenates. Means ± SEM are given; n�10in each group. **P � 0.01.

T3, BAX, AND APOPTOSIS IN XENOPUS TADPOLES 805

muscle fibers, and only in muscle fibers (7; Fig. 3A).

Similarly, as shown in Fig. 3B, at 24 h postinjection

(p.i.) with 1 .tg pcDNA3-Bax, luciferase activity washalf that of the values in control tadpoles injectedwith luciferase and an irrelevant plasmid (GFP). At

48 h p.i., levels in tissue extracts from Bax-injectedanimals were reduced by 80% compared to controls,and decreased to 6% of control values at 72 h p.i.

(Fig. 3B). In controls injected with irrelevant plasmidand luciferase, luciferase expression did not decreaseover time. This shows that overexpressing protein inthe tail muscle does not modif’ muscle fiber survival.

To verify that injection of a pcDNA3-Bax expressionvector resulted in production of Bax mRNA and de-termine whether it was followed by apoptosis, we usedanatomically matched sections for ISH and TUNEL.As shown in Fig. 4, injection of 1 j.tg pcDNA3-Bax plas-mid into the dorsal muscle resulted 24 h later in ex-pression of Bax mRNA around the site of injection(Fig. 4B), whereas injection of a control plasmid pro-duced no Bax signal (Fig. 4A). Bax mRNA productionwas correlated with apoptosis because numerous

apoptotic nuclei were found around the site ofpcDNA3-Bax injection (Fig. 4D), whereas no apoptoticnuclei were found in animals injected with controlplasmid (Fig. 4C). Similar images of Bax mRNA ex-pression and apoptosis were seen at 48 h p.i. (data

not shown).

T3 exerts transcriptional effects on Baxtranscription in a spatially defined manner

To explore the possibility that T3 could regulate tran-

scription of the endogenous Bax gene, a human Baxpromoter-CAT construct (pTM667-3; 19) was in-

jected into the dorsal or caudal tail muscles. As shown

in Fig. 5A, expression was low in dorsal muscle at 2and 5 days p.i.. To ensure that the heterologous pro-moter was functional in Xenopus dorsal muscle, we

coinjected it with a Xenopus p53 expression vector, asp53 is known to activate Bax transcription in othersystems (19). A 30-fold increase in Bax transcriptionresulted, which was unaffected by T3. To check that

the lack of T3 effects on Bax transcription was not dueto lack of T3 receptors (TRs), a Xen opus TRf expres-sion vector was coinjected with pTM667-3. However,this did not affect Bax transcription, with or withoutT3 in dorsal muscle (Fig. 5A). In contrast, we foundthat measurable CAT activity from pTM667-3 and T3

significantly increased Bax transcription in the caudalmuscle after 5 days (Fig. 5B).

T3 exacerbates the apoptotic effect of exogenousBax in dorsal muscle without inducing endogenousBax expression or altering Bax protein stability

To examine whether T3 affected apoptosis after over-expression of Bax, we coinjected pcDNA3-LUC witheither a control plasmid (pcDNA3) or pcDNA3-Baxinto dorsal muscle of control and treated animals. T3treatment significantly reduced by 60% (P�O.05) thelevels of luciferase expressed with pcDNA3-Bax,whereas T3 had no effect on luciferase expression in

animals injected with control plasmid (Fig. 6A).This raised the question of whether T3 was acting

by stabilizing Bax protein. Thus, we followed murineBax protein levels in muscles of murine Bax-injectedcontrol and T3-treated tadpoles. Levels of a 21 kDaprotein were equivalent in both experimental con-ditions (Fig. 6B, lanes 3 and 4). That the 21 kDa pro-

Figure 4. Transfection of pcDNA3-Bax intothe dorsal muscle induces Bax mRNA ex-pression and apoptosis 24 h postinjection.Using sequential cryostat sections of tailmuscle, Bax mRNA expression (A, B) wasfollowed by ISH. Apoptosis (C, D) was fol-lowed using the TUNEL technique. A, C)A region of dorsal muscle from a controlanimal injected with 1 jig of an empty vec-tor showing background hybridization sig-nal (A) and no stained apoptotic nuclei(C). B, D) Area of dorsal muscle from atadpole injected with 1 jig pcDNA3-Baxshowing a dense Bax signal (B) and nu-merous apoptotic nuclei labeled red by al-kaline phosphatase (D, short, thickarrows). x3000. Thin arrows indicate thenormal nuclei stained with hematoxylin.

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806 Vol. 11 August 1997 The FASEB Journal SACHSET AL.

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Time of Control Control TR13-treatment: 48 h 5 days 48 h

B

FigureS. T3 treatment induces transcription from the Bax pro-moter when introduced into the caudal tail muscle but notthe dorsal muscle. A) T treatment is without effect on tran-scription from the human Bax promoter in dorsal muscle. 1jig of pTM 66 7-3 containing the human Bax promoter and0.1 jig of PcDNA3-LUC, for normalization, were injected intothe dorsal muscle either alone or with an expression vectorfor Xenopus TRrI (1 jig) or Xenopus p53 (1 jig). Note that tran-scription is measurable only in the presence of p53 and thatT treatment, whether for 48 h or 5 days, is always withouteffect. B) Five days of T3 treatment activates transcription fromthe human Bax promoter in caudal muscle. *P � 0.05. Means± SEM are given; n � 10 in all cases. The drawing representsa st54 tadpole with the sites of transfection indicated.

tein was murine Bax is supported by the fact that theband is absent in tadpoles injected with an irrelevantplasmid (Fig. 6B, lane 1) and that preabsorbing theanti-Bax antibody with a Bax peptide eliminates thesignal (Fig. 6B, lane 2).

DISCUSSION

There is a consensus that levels of the Bcl-2/Bax genefamily products provide a checkpoint in regulatingcell survival and death in various species and withindifferent tissues. In particular, Bcl-2 and Bcl-xL areable to prevent apoptosis in various systems, whereas

Bax usually counters their effects and accelerates

death (12, 15, 26). However, some exceptions to this

rule have been described (see, for instance, ref 27).Even though it is not certain whether Bax-Bax homo-

dimers form the active component or that heterodi-mers interface with the death effector program, Bax

levels are thought to be critical in apoptosis

#{149}- T3 (28, 12).o + T3 Two observations described here support the hy-

pothesis that Bax exerts death-promoting activity invivo. First, we show that Bax expression and apoptosisare correlated during naturally occuring metamor-phosis and T3 treatment; second, production of Bax

protein by gene transfer results in apoptosis. The reg-

ulation of Bax expression during metamorphosis cor-roborates the hypothesis of Cruz-Reyes and Tata (29)that Bax and not Bc12-like genes are regulated duringtail regression. These authors cloned two Xenopus

Figure 6. A) Detection of the killing activity of Bax by lucif-erase cotransfection and its exacerbation by T3 treatment inthe dorsal muscle. Tadpoles were cotransfected in the dorsalmuscle with I jig luciferase marker plasmid and either 1 jig

of a control plasmid (vector) or a plasmid-expressing mouseBax. Immediately after injection, half of the animals were T5-treated for 48 h, after which all animals were killed. Luciferasewas assayed in muscle homogenates. Means ± SEM are given;nalO in each group. *� 0.05, **J) 0.01. B) T3 treatment

does not modify the stability of exogenously expressed Baxprotein in dorsal muscle of Xenopus tadpoles. Western blottingwas used to follow Bax expression in animals that had beeninjected 24 h earlier with 1 jig pcD NA 3-B ax and then exposedto T3 treatment or left as untreated (controls). Arrow indicatesthe 21 kDa band present in all Bax-injected animals (controlsand T3-treated) but absent in muscle injected with an emptyplasmid vector (lane 1); the band is not detected in Bax-in-jected muscle when a Bax peptide-preabsorbed antibody isused for immunoprecipitation (lane 2).

T3, BAX, AND APOPTOSIS IN XENOPUS TADPOLES 807

Bc12 genes and examined their expression duringmetamorphosis. No modification of expression oc-

curred in the regressing tail, so the authors suggested

apoptosis might implicate regulation of a death-pro-moting gene.

Although there is agreement that Bax/Bcl-2equilibrium is intimately involved in cell deathregulation, two questions remain: how changes inBcl-2/Bax homeostasis are integrated at the nu-clear level and whether extracellular or intracel-lular signals can stimulate apoptosis without going

through the Bcl-2/Bax gate. Our data shed lighton the second question. They suggest that extra-cellular signals inducing cell death can synergize

with the Bax signal to accelerate the death-pro-moting machinery without actually modulatingBax levels. Indeed, when exogenous Bax is ex-pressed in the dorsal muscle, T3 exacerbates apop-tosis independently of endogenous Bax activation.Moreover, T3 does not activate transcription froma Bax promoter transferred into dorsal muscle,though the same promoter is functional in thecaudal muscle. Also, T3 does not induce endoge-nous Bax mRNA in the dorsal muscle within thetime frame used. Finally, Western blotting showsthat levels of murine Bax produced from the trans-

gene in the dorsal muscle are not modulated byT3. So the exacerbation of exogenous Bax-inducedapoptosis by T3 in the dorsal muscle most probablyoccurs independently of endogenous Bax activa-tion or stabilization of Bax.

Two groups of signals activated by T3 could beimpinging on the cell death program: direct acti-vation of caspases in the same cell or indirect ac-tivation by extracellular enzymes. Recently, Yaoita

and Nakajima (30) have shown that T3-inducedapoptosis in a tadpole tail-derived cell line impli-cates the CPP32 caspase. Turning to the origin ofextracellular signals, a good candidate is the epi-dermis. For apopotosis to be induced in musclecells in vitro, epidermal cells must be present (31).

Possible signals arising from the epidermis includemetalloproteinases, of which two-stromelysin 3and collagenase-are up-regulated by T5 in thetadpole tail (32). Thus, T3 treatment could causeremodeling of the extracellular matrix (ECM),then induce changes in cell/cell or cell/ECMcontacts, which in turn activate the apoptoticprogram. Indeed, it is known that ECM contactis a survival factor involving integrin signal-

ing (33).In conclusion, these results obtained in an inte-

grated, in vivo system provide evidence that Bax ex-pression is associated with apoptosis during naturalmetamorphosis and that overexpressing the protein

by somatic gene transfer can induce the apoptoticprocess. Future work on this model will allow us tofurther dissect the molecular cascade of events lead-

ing to apoptosis and tail regression in a physiologi-cally appropiate context.

We thank the Association Francaise contre les Myopathies,the Association pour Ia Recherche contre le Cancer, theCNRS, and the MNHN for financial support, and Dr. C.Jouanin and Dr. M. Laurent (Service d’imagerie scientifique,Universit#{233}de Paris XI) for help in quantifying the in situ hy-bridization. Gerard Benisiti and Etienne Le Goff provided an-imal care. L.M.S. was a recipient of Minist#{232}rede Ia Rechercheand the Ligue Nationale contre le Cancer scholarships. G.L.was supported by Telethon Italy (project D33) and AIRC.

REFERENCES

1. White, E. (1996) Life, death, and the pursuit of apoptosis. Genes&Dev. 10, 1-15

2. Raff, M. C. (1992) Social controls on cell survival and cell death.Nature (London) 356, 397-400

3. Kerr,J. F. R., Harmon, B., and Searle,J. (1974) An electron-microscope study of cell deletion in the Anuran tadpole tail dur-ing spontaneous metamorphosis with special reference toapoptosis of striated muscle fibres. J. Cell Sri. 14, 57 1-585

4. Tata, J. R. (1993) Gene expression during metamorphosis: anideal model for post-embryonic development. BioEssays 15,239-248

5. Shi, Y.-B., Wong,J., Puzianowska-Kuznicka, M., and Stolow, M.A.(1996) Tadpole competence and tissue-specific temporal regu-lation of amphibian metamorphosis: roles of thyroid hormoneand its receptors. BwEssays 18, 391-399

6. Tata, J. R. (1994) Hormonal regulation of programmed celldeath during amphibian metamorphosis. Biochem. Cell Biol. 72,581-588

7. de Luze, A., Sachs, L., and Demeneix, B. (1993) Thyroid hor-mone-dependent transcriptional regulation of exogenous genestransferred into Xenopus tadpole muscle in vivo. Pox. Natl. Acad.Sri. USA 90, 7322-7326

8. Sachs, L., de Luze, A., Lebrun, J.-J., Kelly, P. A., and Deme-neix, B. A. (1996) Use of heterologous DNA-based gene trans-fer to follow physiological, T3-dependent regulation ofmyosin heavy chain genes in Xenopus tadpoles. Endocrinol.137, 1291-1294

9. Werner, M. H. (1996) Stopping death cold. Structure4, 879-88310. Farrow, S. N., and Brown, R. (1996) New members of the bcl-2

family and their protein partners. Cur,-. Opin. Genet. Den. 6, 45-49

11. Hockenbery, D. M. (1995) bcl-2, a novel regulator of cell death.BioEssays 17, 631-638

12. Knudson, C. M., lung, K. S. K., Tourtellotte, W. G., Brown,G. A. J., and Korsmeyer, S. J. (1995) Bax.deficient mice with lym-phoid hyperplasia and male germ cell death. Science 270, 96-99

13. Nieuwkoop, P. 0., and Faber,J. (1975) Normal Table of Xenopuslaevis (Daudin), 2nd Ed, North-Holland, Amsterdam

14. Sambrook,J., Fritsch, E. F., and Maniatis, T. (1989) MolecularCloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Lab.Press, Plainview, New York

15. Oltvai, Z. N., Milliman, C. L., and Korsmeyer, S.J. (1993) Bcl-2heterodimerizes in vivo with a conserved homolog, Bax, that ac-celerates programed cell death. Cell 74. 609-619

16. Heim, R., Cubitt, A. B., and Tsien, R. V. (1995) Improved greenfluorescence. Nature (London) 373, 663-664

17. Soussi, 1., Caron de Fromentel, C., Mechali, M., May, P., andKress, M. (1987) Cloning and characterization of a cDNA fromXenopus laevis coding for a protein homologous to human andmurin p53. Oncogene 1, 71-78

18. Machuca, I., Esslemont, G., Fairclough, L., and Tata,J. R. (1995)Analysis of structure and expression of the thyroid hormone re-ceptor- gene to explain its autoinduction. Mol. Endocrinol. 87,105-113

19. Miyashita, T., and Reed. J. C. (1995) Tumor suppressor p53 is adirect transcriptional activator of the human bax gene. Cell 80,293-299

808 Vol. 11 August 1997 The FASEB Journal SACHS EF AL.

20. Whitfield, H. J., Brady, L. S., Smith, M. A., Mamalaki, E., Fox,R.J., and Herkenham, M. (1990) Optimization of cRNA probein situ hybridization methodology for localization of glucocor-ticoid receptor mRNA in rat brain: A detailed protocol. Cell. Mol.Neurobiol. 10, 145-157

21. Krajewski, S., Krajewska, M., Shabaik, A., Miyashita, T., Wang,H. G., and Reed, J. C. (1994) Immunohistochemical determi-nation of in vivo distribution of Bax, a dominant inhibitor of Bcl-2. Am.]. Pathol. 145, 1323-1333

22. Miyashita, T., Kitada, S., Krajewski, S., Home, W. A., Delia,D., and Reed,J. C. (1995) Overexpression of the Bcl-2 proteinincreases the half-life of p21’.]. Biol. Che,n. 270, 26049-26052

23. Gavrieli, V., Sherman, V., and Ben-Sasson, S. A. (1992) Identifi-cation of programmed cell death in situ via specific labeling ofnuclear DNA fragmentation.]. Cell Bud. 119, 493-501

24. Leloup, J., and Buscaglia, M. (1977) La triiodothyronine: hor-mone de Ia metamorphose des amphibiens. C. It Acad. Sci. Paris284, 2261-2263

25. Chittenden, T., Hemington, C., Houghton, A. B., Ebb, R. G., Gallo,G.J., Elangovan, B., Chinnadurai, G., and Lutz, R.J. (1995) A con-served domain in Bak, distinct from BH1 and BH2, mediates celldeath and pontein binding functions. EIriBOJ 14, 5589-5596

26. Oltvai, Z. N., and Korsmeyer, S.J. (1994) Checkpoints of duelingdimers foil death wishes. Cell 79, 189-192

27. Middleton, G., Nunez, G., and Davies, A. M. (1996) Bax pro-motes neuronal survival and antagonises the survival effects ofneurotrophic factors. Development 122, 695-701

28. Yang, E., Zha,J., Jockel,J., Boise, L. H., Thompson, C. B., andKorsmeyer, S.J. (1995) Bad, a heterodimeric partner for Bcl-xLand Bcl-2, displace Bax and promotes cell death. Cell 80, 285-291

29. Cruz-Reyes, J., and Tata, J. R. (1995) Cloning, characterizationand expression of two Xenopus bcl-2-like cell-survival genes. Gene158, 171-179

30. Yaoita, V., and Nakajima, K. (1997) Induction of apoptosisand CPP32 expression by thyroid hormone in a myoblasticcell line derived from tadpole tail. J. Biol. Chem. 272, 5 122-5127

31. Niki, K., Namiki, H., Kikuyama, S., and Yoshizato, K. (1982) Ep-idermal tissue requirement for tadpole tail regression inducedby thyroid hormone. Den. Biol. 94, 116-120

32. Wang, Z., and Brown, D. D. (1993) Thyroid hormone-inducedgene expression program for amphibian tail resorption. J. BioLC/tern. 268, 16270-16278

33. Ruoslahti, E., and Reed, J. C. (1994) Anchorage dependence,integrins, and apoptosis. Cell 77, 477-478

Received for publication April 1, 1997.Accepted for publication May 30, 1997.