PATTERNS & PHENOTYPES

Localization of Apaf1 Gene Expression in theEarly Development of the Mouse by Means ofIn Situ Reverse Transcriptase-PolymeraseChain ReactionMatthias Muller,1,2* Joachim Berger,3 Nikolaus Gersdorff,2 Francesco Cecconi,4 Rainer Herken,1

and Fabio Quondamatteo1

Apoptosis is an essential ubiquitous process that controls the duration of the life span of cells, thus playing acrucial role in morphogenetic, histogenetic, and phylogenetic developmental processes. Apaf1 (apoptosisprotease activating factor 1) is one of the central mediators of the intrinsic apoptotic pathway and a part of theapoptosome, which activates procaspase-3 and promotes cell death. Gene knockout of Apaf1 in mice leads to lateembryonic lethality with malformations such as the persistence of interdigital webs and hyperplasia of brainand retina. Therefore, Apaf1 is generally believed to play a crucial role in developmental apoptosis and have awidespread expression. However, its pattern of expression in early development remains unknown. To specifywhether Apaf1 indeed plays this key role, we investigated the pattern of gene expression for Apaf1 in mouseembryos on day 7, 9, and 12 of development. Our results show, that gene expression for Apaf1 first occurs withinthe embryo between day 7 and 9 of development, becoming more widespread toward day 12 and then includesstructures, such as yolk sac, mesenchyme, cartilage, heart anlage, otic vesicle, peridermis, and anlagen of thespinal ganglia and vertebral bodies. Our results also show that gene expression for Apaf1 is not ubiquitous inearly mouse development. This finding indicates that cell death processes are independent of or less dependenton Apaf1 during this time. Of interest, an active gene expression for Apaf1 is also present in organ anlagen suchas heart or intestine, in which no obvious phenotype is seen after Apaf1 deletion. This finding suggests apossible role for Apaf1 in such anlagen as a putative alternative compensatory pathway, which could beswitched on in the case of defects in the mediators that are normally involved in such organs. DevelopmentalDynamics 234:215–221, 2005. © 2005 Wiley-Liss, Inc.

Key words: apoptosis; early development; Apaf1; in situ RT-PCR

Received 19 April 2005; Revised 3 June 2005; Accepted 16 June 2005

INTRODUCTION

Apoptosis, i.e., programmed cell death(PCD), is a process which controls theduration of the life span of cells in

several adult and developing tissues(Kerr et al., 1972; Jacobson et al.,1997). This process is essential for thephysiological control of developmentand of the homeostasis of adult tis-

sues. In early mouse embryos, for ex-ample, the preamniotic cavity isformed by PCD of ectodermal cells inthe core of the developing embryo.Also epithelial invagination and for-

1Department of Histology, University of Gottingen, Germany2Department of Prosthodontics, University of Gottingen, Germany3Department of Molecular Biology, MPI for Biophysical Chemistry, Gottingen, Germany4Dulbecco Telethon Institute, Department of Biology, University Tor Vergata, and IRCCS Fondazione Santa Lucia–CERC, Rome, ItalyGrant sponsor: Telethon; Grant number: TCP99038; Grant sponsor: AIRC, M.IUR/FIRB; Grant number: RBAU01FZMZ; Grant sponsor: theCompagnia di San Paolo.*Correspondence to: Matthias Muller, Department of Prosthodontics, University of Gottingen, Robert-Koch-Stra�e 40,D-37075 Gottingen, Germany. E-mail: mmuelled@gwdg.de

DOI 10.1002/dvdy.20534Published online 5 August 2005 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 234:215–221, 2005

© 2005 Wiley-Liss, Inc.

mation of tubes and vesicles as well asthe formation of the vertebrate neuraltube or lens are strictly dependent onPCD. As organs develop, many cellsare produced in excess and then re-moved by PCD. In the developing ner-vous system, for example, neuronsand oligodendrocytes are overpro-duced and up to 50% are eliminated infollowing steps. Under the influence ofPCD, there are also deletions of vesti-gial structures that are required inancestral species but not in the de-scendant, or the removal of structuresthat are needed in one sex but not inthe other. The Mullerian duct, for ex-ample, which is only needed in femalemammals because it forms the uterusand the oviducts is eliminated by PCDin males during development. The im-portance of apoptosis during develop-ment, for example, has been demon-strated in several gene knockouts, forexample, in caspase-3 knockout, inwhich it leads to late embryonic le-thality and several resulting malfor-mations during embryogenesis (Ja-cobson et al., 1997).

Apaf1 (apoptosis protease activat-ing factor 1) is a mediator of the in-trinsic apoptotic pathway. Togetherwith activated caspase-9 and cyto-chrome c, Apaf1 is a part of the apop-tosome. This holoenzyme is often con-sidered to be the core mediator ofapoptosis. It indeed activates pro-caspase-3 and, thus, plays a centralrole in apoptotic processes (Zou et al.,1997; Rodriguez and Lazebnik, 1999;Li, 1997; Cain, 2000). Lack of Apaf1 inmice has also been found to be respon-sible for a late embryonic lethality.Embryos deficient in Apaf1 show sev-eral malformations such as the persis-tence of interdigital webs, overgrowthof brain, retina, and inner ear defects(Cecconi et al., 1998, 2004). The geneexpression for Apaf1 is generally be-lieved to be ubiquitous, although doc-umented proof is still lacking. Despitethe suggested importance of this me-diator in development, it remains un-known whether it really takes part inPCD in all kinds of tissues. That sucha marked neural phenotype is presentwhen defects in Apaf1 occur, indicatesthat Apaf1 is expressed in neural tis-sues during development. By means ofin situ hybridization, mRNA for Apaf1was shown to be present in brains andthe developing inner ear of 9.5-day-old

mouse embryos (Cecconi et al., 1998;Yoshida et al., 1998). Also, data fromwhole mount embryos on the activa-tion of the reporter gene lacZ in braintissue of the Apaf1�/� mouse embryoindirectly indicate expression of thismediator in neural tissues (Cecconi etal., 2004).

In contrast, nothing is known on thelocalization of gene expression forApaf1 in early developmental stagesin the non-neural tissues. In 1997,Northern blot analysis of numerousadult human organs demonstratedgene expression for Apaf1 (Zou et al.,1997). This circumstance and the es-timated central role that Apaf1 couldplay in apoptosis during adulthoodand development gave rise to the ideathat Apaf1 could be expressed ubiqui-tously in adult and particularly in de-veloping individuals even in non-neu-ral tissues (Glucksmann, 1951; Zou etal., 1997; Cecconi et al., 1998). How-ever, this has yet to be elucidated.Therefore, in the present work, we in-vestigated the localization of gene ex-pression for Apaf1 in early embryonicdevelopment of the mouse on day 7, 9,and 12 of gestation. We chose to use insitu reverse transcriptase-polymerasechain reaction (RT-PCR), which due tothe amplification of the signal, is ahighly sensitive method (Bagasra etal., 1992; Nuovo et al., 1992; Peters etal., 1997; Miosge et al., 2002). Ourresults should help to elucidatewhether Apaf1 is also present and,therefore, may function in non-neuraltissues as part of the core of PCD dur-ing development.

RESULTS AND DISCUSSION

In the present work, we aimed toinvestigate gene expression forApaf11 in early mouse developmentwith particular emphasis on non-neural tissues. Whereas numerousdata indicate expression of Apaf1 inneural tissues in development (Cec-coni et al., 1998; 2004; Yoshida et al.,1998), there is hardly any informa-tion on expression of this apoptosismediator in non-neural tissues dur-ing early development. We used insitu RT-PCR to localize transcriptsfor Apaf1 in mouse development ondays 7, 9, and 12, i.e., time periods inwhich several developmental stepsoccur and active apoptotic processes

are expected. The advantage in us-ing in situ RT-PCR is that it allowsdetection of transcripts in tissue sec-tions with high sensitivity. In fact,due to the amplification of the se-

Fig. 1. Gel electrophoresis of polymerasechain reaction (PCR) products after amplifica-tion of murine embryonic mRNA probes bymeans of in vitro reverse transcriptase-PCR us-ing primers specific for Apaf1 (lane 2), forglyceraldehyde-3-phosphate dehydrogenase(GAPDH; lane 3, positive control) and by omit-ting the primers (lane 4, negative control). Aband of approximately 630 base pairs is visiblein lane 2, corresponding to the size of the PCRproduct given by the primer pair used. A bandof approximately 450 base pairs is visible inlane 3, corresponding to the size of the PCR-product given by the primer pair used. No spe-cific band is visible in lane 4. The low base pairsmears in lanes 3 and 4 may represent nonam-plified template and primer residues.

Fig. 3. A: Mouse embryo day 12. Detection ofApaf1 gene expression in the heart anlage (ar-rows). Scale bar � 45 �m. B: Mouse embryoday 12. Detection of Apaf1 gene expression inthe yolk sac (arrows). Scale bar � 40 �m.C: Mouse embryo day 12. Detection of Apaf1gene expression in hyaline cartilage cells (ar-rows). Scale bar � 40 �m. D: Mouse embryoday 12. Detection of Apaf1 gene expression inprimitive intestine (arrows). Scale bar � 40 �m.E: Mouse embryo day 12. Detection of Apaf1gene expression in the anlage of a spinal gan-glion (arrows). Scale bar � 60 �m. Inset: Highermagnification of the same object. Detection ofApaf1 gene expression (arrows). Scale bar � 30�m. F: Mouse embryo day 12. Detection ofApaf1 gene expression in the peridermis in theregion of invagination of the otic vesicle (ar-rows). Scale bar � 85 �m. Inset: Higher mag-nification of the same object. Detection ofApaf1 gene expression (arrows). Scale bar � 35�m.

216 MULLER ET AL.

quence approximately 1012 times ina performance of 40 cycles, very lowamounts of mRNA can be specificallydetected in the tissue (Bagasra et al.,

Fig. 3.

Fig. 2. A: Mouse embryo at day 7. No geneexpression for Apaf1 is detectable in the em-bryo. ex, extraembryonic tissue; asterisk,endoderm; d, decidua; ect, ectoderm; m, me-soderm. Scale bar � 150 �m. Inset: Detectionof Apaf1 gene expression in decidua cells (ar-rowhead) from the same section of the embryoas depicted in Figure 2A. Scale bar � 20 �m. B:Mouse embryo day 9. Detection of Apaf1 geneexpression in the heart anlage (arrows). Scalebar � 30 �m. C: Mouse embryo day 9. Detec-tion of Apaf1 gene expression in primitive intes-tine (arrows). Scale bar � 30 �m. D: Mouseembryo day 9. Detection of Apaf1 gene expres-sion in the yolk sac (arrows). Scale bar � 20�m.

Apaf1 EXPRESSION IN EARLY DEVELOPMENT 217

1992; Nuovo et al., 1992; Peters etal., 1997; Miosge et al., 2002). Byusing digoxigenin (DIG) -11–labeleddUTP instead of dTTP in the label-ing step of the in situ RT-PCR, animmunohistochemical detection ofthe processed amplificate with alka-line phosphatase– conjugated anti-DIG antibodies is possible.

A major problem in using this tech-nique is that, due to aggressive re-agents such as proteinase K and tem-perature changes between 4°C and94°C, only a limited preservation ofhighly sensitive tissues such as brainor the developing neural tube is possi-ble. However, because it was our pri-mary aim to describe Apaf1 expres-sion in extraneural tissues and alsogiven the limited preservation of neu-ral tissues after the in situ PCR reac-tions, we excluded these latter tissuesfrom the evaluation of the pattern ofdistribution of the transcripts forApaf1.

On day 7, no expression of mRNAspecific for Apaf1 was detected withinthe embryo (Fig. 2A). However, ex-pression for Apaf1 was seen in thedecidual cells (of maternal origin)within the same sections (Fig. 2A. in-set).

Previously, it was shown by North-ern blots from whole mouse embryosthat no gene expression of Apaf1 wasdetectable anywhere in the embryo onday 7.5 (Cecconi et al., 1998). Thisfinding is in line with our findings,and we can now confirm the absence ofApaf1 expression in 7-day-old mouseembryos on the cellular level bymeans of in situ RT-PCR.

Whereas embryonic tissue did notshow any Apaf1 expression, tran-scripts for Apaf1 were specifically de-tected in the surrounding deciduacells. This finding suggests that thelack of staining of embryonic struc-tures was not due to technical mis-takes but indeed to still unstimulatedgene expression for Apaf1. Therefore,this is a useful internal positive con-trol, and we can conclude that, on day7, no detectable expression for Apaf1is present in the embryo.

Moreover, decidua cells have beenshown to express other mediators ofthe intrinsic apoptotic pathway suchas caspase-3, caspase-9, Bax, and Bcl2(Joswig et al., 2003). To these estab-lished results, we can now add that

Apaf1 is also expressed in the decidualcells. This observation strongly speaksfor the activation of the intrinsicpathway of PCD in decidua cells.

In 1995, investigations from Cou-couvanis and Martin indicated thatcavitation processes of the mouse em-bryo, which take place between day 5and 7 of embryogenesis, show hall-marks of PCD such as DNA fragmen-tation (Coucouvanis and Martin,1995). Our results now indicate that,in such early developmental stages,apoptotic processes may be primarilyinfluenced by other pathways and me-diators, because Apaf1 was not detect-able within the embryo. PCD, which isresponsible for cavitation of the earlymouse embryo, for example, was latershown to strongly depend on bonemorphogenetic protein (BMP)-2 and -4docking on death receptors, which,similarly to the extrinsic pathway, ac-tivate an intracellular cascade depen-dent on apoptosis-inducing factor(AIF). Knockout of either AIF orBMP-2 and -4 prevents cavitation inembryoid bodies (Coucouvanis andMartin, 1999; Joza et al., 2001). Thelack of expression for Apaf1 on day 7,as shown in the present work, mightexclude a possible role of Apaf1 duringthis time under normal conditions.

On day 9, expression for Apaf1mRNA was detectable in the heart an-lage, in the periderm, in the primitiveintestine, and in mesenchymal cells,as well as in the yolk sac (Fig. 2B–D).Also on day 12, expression of mRNAspecific for Apaf1 was seen in the yolksac (Fig. 3B) and in several other em-bryonic structures. Herein, gene ex-pression for Apaf1 was clearly seen inthe mesenchyme, in cartilage (Fig.3C), in the heart anlage (Fig. 3A), inthe periderm, in the primitive intes-tine (Fig. 3D), in the anlagen of thespinal ganglia (Fig. 3E), otic vesicle(Fig. 3F), and in the anlagen of thevertebral bodies.

Whole-mount hybridization in mouseembryo showed gene expression forApaf1 on day 9.5 (Yoshida et al., 1998).From our study, we can show that geneexpression for Apaf1 in murine develop-ment already begins between day 7 and9 in heart, mesenchyme, periderm, andprimitive intestine.

We showed that, on day 12, Apaf1expression became more widespread,then including other organ anlagen

and tissues such as cartilage, spinalganglia, and vertebral bodies. Apaf1was believed to be expressed ubiqui-tously in vertebrates since its expres-sion was demonstrated in severaladult human tissues (Zou et al., 1997).We now show that, during develop-ment, Apaf1 is not expressed ubiqui-tously and, therefore, cannot take partin PCD in all tissue types. The ab-sence of expression for Apaf1 in someof the organ anlagen or in some tis-sues, on the other hand, does not ex-clude the presence of apoptosis. Al-though generally protected againstapoptotic stimuli, embryonic stem celland fibroblast knockouts for the Apaf1gene still retain some apoptotic poten-tial, for example after ultraviolet lightstimulation (Yoshida et al., 1998).Similarly, CD4-positive and CD8-pos-itive T-immunoblasts as well as B-im-munoblasts deficient in Apaf1 orcaspase-9 are not protected againstcell death in the absence of cytokines(Marsden et al., 2002). This findingsuggests that apoptosis can take placewithout Apaf1 and that the influenceof Apaf1 in directing PCD can vary,depending on the cell type and on theindividual stimuli.

Earlier reports demonstrated thatmitochondria could contain mediatorsdownstream from Apaf1, such as pro-caspase-9 or procaspase-3. Possibly, dif-ferent proapoptotic stimuli, for exam-ple, oxidative stress, may inducecaspase autoprocessing and release ofcaspases into the cytosol. In this way,under certain conditions, by shuntingApaf1 and formation of the apoptosome,Apaf1-independent but caspase-depen-dent apoptotic processes could takeplace (Katoh et al., 2004).

It is also possible that, instead ofApaf1, other mediators may be able toact pro-apoptotic in the intrinsic path-way when Apaf1 is not expressed.Some proteins with structures verysimilar to Apaf1 have been described.Nac/Defcap (NBD and CARD/deatheffector filament forming ced-4-likeapoptosis protein), for instance, con-sists of an NBD (nucleotide bindingdomain) and a CARD (caspase recruit-ment domain) as well as Apaf1. TheCARD domain enables Nac/Defcap tobind caspase-9 and caspase-2. Be-cause of the structural similarities toApaf1, also functional similarities canbe postulated (Chu et al., 2000; Hlaing

218 MULLER ET AL.

et al., 2001). Nod1 and Nod2 also con-sist of a CARD and an NBD and,therefore, share structural similari-ties with Apaf1. Whereas the functionof Nod2 is still unclear, Nod1, despitethe structural similarity, has beenfound to be functionally different fromApaf1 (Inohara et al., 1999, 2001).However, the presence of severalstructurally related proteins may gen-erally speak for some of them beingable to accomplish functions similar toApaf1.

Another process promoting celldeath during development in vivo in atotal caspase- or Apaf1-independentmanner, could be necrotic cell death.In fact, the presence of necrotic cellshas been reported in interdigital websin vivo after blocking of caspase activ-ity or knockout of Apaf1 (Chautan etal., 1999). Similar mechanisms mayapply in early embryonic stages in theabsence of Apaf1. Alternatively, lowApaf1 expression present in earlystages under the limits of detection oran expression of Apaf1 inducible uponpro-apoptotic stimuli but not presentunder basal conditions may accountfor cell death in early embryos.

Furthermore, one could speculatethat, in tissues in which no Apaf1 isexpressed, apoptotic processes even inlater developmental steps may pre-dominantly occur by means of shed-ding of BMPs with activation of deathreceptors and of the AIF pathway orby means of activation of the classicextrinsic pathway. However, whatcells might be responsible for the pro-duction of signal molecules responsi-ble for the activation of death recep-tors in development remains unclear.

Our results additionally show thatApaf1 is expressed in organ anlagenand at time points in which no malfor-mations have been detected after de-letion of Apaf1 in vivo. In fact, inApaf1 knockouts, in addition to inter-digital webs, the most evident pheno-typical manifestations are present inneural tissues such as brain, spinalcord, retina, and inner ear anlage(Cecconi et al., 1998). There is, indeed,in vitro evidence for apoptosisstrongly dependent on Apaf1 in cellsof neural origin (Cozzolino et al.,2004), whereas other non-neural cellsare less dependent thereon (Yoshidaet al., 1998; Marsden et al., 2002). Invivo, no obvious manifestations of the

phenotype have been found in organanlagen such as heart, primitive in-testine, or skin (Cecconi et al., 1998;Yoshida et al., 1998) in which Apaf1now has been shown to be expressedduring development. This, in turn,could be a further clue for the exis-tence of some of the mediators in ad-dition to Apaf1 throughout the intrin-sic pathway or other caspase-dependent or -independent pathwaysthat lead to PCD as noted above. Onthe one hand, it is possible that, insuch organs, Apaf1-mediated PCDnormally occurs during developmentbut, after its deletion, Apaf1 functionsmay be otherwise compensated for.On the other hand, Apaf1 could alsobe present in the tissue as a part of aputative alternative pathway, whichin the absence of the mediators thatare otherwise normally active, can beactivated in these organ anlagen.

Conclusions

In the present work, we have demon-strated the localization of gene expres-sion for Apaf1 in early mouse develop-ment. Our results showed that geneexpression for Apaf1 begins betweenday 7 and 9 and that it is not ubiqui-tous in early mouse development. Thisfinding suggests that cell death pro-cesses are independent of or less de-pendent on Apaf1 during this time. Ofinterest, an active gene expression forApaf1 is also present in organ anlagensuch as heart or intestine, in which noobvious phenotype is seen after Apaf1deletion. This finding may suggest apossible role of Apaf1 in such anlagenas a putative alternative compensa-tory pathway that could be switchedon in the case of defects in the media-tors that are normally involved insuch organs.

EXPERIMENTALPROCEDURES

Tissue Studied

Pregnant NMRI mice were killed bycervical dislocation on days 7, 9, and12 post coitum, and the uteri wereimmediately removed. The day 7 em-bryos were fixed in toto with the cor-responding uteri for 24 hr at 4°C in 4%phosphate buffered formaldehyde.Day 9 and day 12 embryos were first

isolated from the uteri in HTK-solu-tion (Quondamatteo et al., 1994) and,thereafter, fixed in 4% phosphate-buffered formaldehyde for 24 hr at4°C. After having been washed in 70%ethanol, the tissue specimens weredehydrated in an ascending ethanolseries and embedded in paraffin ac-cording to our standard protocols(Quondamatteo et al., 2000). Five-mi-crometer-thick sections were then col-lected on SuperFrost Plus slides un-der RNAse free conditions and driedfor at least 12 hr at 55°C.

Primers

Primers corresponding to coding se-quences for the exon 4, which is basenumber 882-900 of murine Apaf1mRNA (sense primer, 5�-AAG GACAGT GCT GTG TGA A-3�) and for theexon 7, which is base number 1488-1508 of murine Apaf1 mRNA (anti-sense primer: 5�-CCT TTG CAT TCCTTT ATA ATA C-3�) were used (acces-sion no. NM_009684, Locus ID 11783).The cDNA sequence amplified by thisprimer pair is 627 base pairs. As apositive control for the in vitro PCRreactions, a standard primer pair cor-responding to the coding sequence formurine glyceraldehyde-3-phosphatedehydrogenase (GAPDH; sense primer,5�-ACC ACA GTC CAT GCC ATCAC-3� and antisense primer, 5�-TCCACC ACC CTG TTG CTG TA-3�) wasused. The GAPDH sense primer corre-sponded to base number 556-575, andthe GAPDH antisense primer corre-sponded to base number 988-1117 ofmurine GAPDH mRNA (accession no.NM_083149, Locus ID 407972). ThecDNA sequence amplified by thisprimer pair is 451 base pairs long.

In Vitro RT-PCR

The optimal working conditions forthe primers specific for the Apaf1 se-quence were tested for specificity by invitro RT-PCR using RNA preparedfrom 18-day-old wild-type NMRImouse embryos. A mix containing (10�l 5� EZ buffer I [Perkin Elmer, Ap-plied Biosystems Foster City, CA] �1.5�l each dATP, dCTP, dGTP,dTTP � 2 �l embryonic mRNA � 5�lMnOac2 [Perkin Elmer] � 2 �l RTthDNA polymerase [Perkin Elmer] � 22�l distilled water) was prepared and

Apaf1 EXPRESSION IN EARLY DEVELOPMENT 219

the primer pairs for the Apaf1 and forG3PDH sequence, respectively, wereadded (1.5 �l of each primer). Nega-tive controls were performed by omit-ting primers. The probes were thenincubated in a thermo-cycler (PerkinElmer) as follows: 1 cycle (40 min at63°C) � 1 cycle (3 min at 94°C) � 40cycles (30 sec at 94°C � 30 sec at57°C � 1 min[in each cycle, the timewas increased by 10 sec] at 68°C) � 1cycle (8 min at 68°C). After cycling, 2�l of each PCR product were takenand added to 3 �l of distilled waterand 1 �l of loading buffer (FMC Bio-medicals, Rockland, ME). Two micro-liters of a 100-bp ladder (FMC Bio-medicals) was added to 3 �l of distilledwater and 1 �l of loading buffer (FMCBiomedicals). Each probe was run in a1.5% agarose gel for 60 min at 100 Vand subsequently visualized withethidium bromide under ultravioletlight (Fig. 1).

In Situ RT-PCR

In situ RT PCR was carried out ac-cording to the method described byMiosge et al. (2002) with slight modi-fications. Briefly, the sections weredeparaffinized, rehydrated, treatedwith proteinase K, and a DNase solu-tion. For the DNAse treatment, thesections were covered with ampli-coverclips and -discs (Perkin Elmer)using the corresponding assembly tool(Perkin Elmer) and incubated for 7 hrat 37°C in the thermocycler (PerkinElmer). Afterward, the clips and discswere removed, and the sections wereincubated in distilled water, then in100% ethanol, and rinsed in TBS. Thesections were then processed for thereverse transcription and amplifica-tion using a mix containing nucleo-tides, RTth DNA polymerase, and thespecific primers (Perkin Elmer). Afterthe application of this mixture, thesections were covered with ampli-coverclips and -discs and processed inthe thermo-cycler as follows: 1 cycle(40 min at 63°C) � 1 cycle (2 min at94°C) � 40 cycles (30 sec at 94°C � 30sec at 57°C � 1 min[in each cycle, thetime was increased by 10 sec] at68°C) � 1 cycle (8 min at 68°C). There-after, the sections were rinsed in TBSand a new mixture containing nucleo-tides, the specific primers, Amplitaqpolymerase (Perkin Elmer), and addi-

tional DIG-11–labeled-dUTP (Boehr-inger, Mannheim, Germany) was ap-plied to the sections. The sectionswere covered again and processed inthe thermo-cycler for one cycle of 3min at 94°C � 2 min at 57°C � 2 minat 68°C. After the cycle, clips and discswere removed, and the sections wereprocessed for the immunohistochemi-cal detection of digoxigenin using al-kaline phosphatase anti-DIG antibod-ies (Boehringer). The immunoreactionwas visualized by incubating the sec-tions for approximately 10 min in thedark with a mixture containing nitroblue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP;Boehringer) and levamisole (Sigma,Munich, Germany). The reactions werethen stopped in TBS, and the sectionswere counterstained with nuclear fastred, dehydrated, and coverslipped. Neg-ative controls were performed either byomitting the RTth DNA polymerase,the primers, or the DIG-labeled dUTPduring the labeling step.

ACKNOWLEDGMENTSWe thank Silke Eckert and PatriciaSprysch for their skillful technical as-sistance. We also thank CyrillaMaelicke for editing the manuscript.F.C. is supported by Telethon, AIRC,M.IUR/FIRB, and the Compagnia diSan Paolo.

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