688 EMBO reports vol. 3 | no. 7 | pp 688–694 | 2002 © 2002 European Molecular Biology Organization
A γ-secretase inhibitor blocks Notch signaling
in vivo and causes a severe neurogenic phenotype
Andrea Geling1,2, Harald Steiner3, Michael Willem3, Laure Bally-Cuif1,2,+&
1Zebrafish Neurogenetics Junior Research Group, Institute for Virology, Technical University Munich, Trogerstrasse 4b, D-81675 Munich,
2GSF-National Research Center for Environment and Health, Institute of Mammalian Genetics, Ingolstaedter Landstrasse 1, D-85764 Neuherberg and
3Adolf Butenandt Institute, Laboratory for Alzheimer’s and Parkinson’s Disease Research, Ludwig Maximilians University Munich, Department of
Biochemistry, Schillerstrasse 44, D-80336 Munich, Germany
Received February 5, 2002; revised April 4, 2002; accepted May 7, 2002
Inhibition of amyloid β-peptide (Aβ) production by blocking
γ-secretase activity is at present one of the most promising
therapeutic strategies to slow progression of Alzheimer’s
disease pathology. γ-secretase inhibitors apparently block Aβ
generation via interference with presenilin (PS) function.
Besides being an essential component of the γ-secretase
complex, PS itself may be an aspartyl protease with γ-secretase
activity, which is not only required for Aβ production but also
for a similar proteolytic process involved in Notch signaling.
Here we demonstrate that treatment of zebrafish embryos
with a known γ-secretase inhibitor affects embryonic develop-
ment in a manner indistinguishable from Notch signaling
deficiencies at morphological, molecular and biochemical
levels. This indicates severe side-effects of γ-secretase inhibitors
in any Notch-dependent cell fate decision and demonstrates
that the zebrafish is an ideal vertebrate system to validate
compounds that selectively affect Aβ production, but not
Notch signaling, under in vivo conditions.
Accumulation of amyloid β-peptide (Aβ) is an invariant feature
associated with Alzheimer’s disease (AD) pathology. Aβ is
generated by the consecutive cuts of two proteases, known as
β- and γ-secretase (Esler and Wolfe, 2001), which liberate the
amyloidogenic peptide from its precursor, the β-amyloid
precursor protein (APP). Subsequent aggregation is thought to
result in the formation of neurotoxic protofibrils (Walsh et al.,
1997; Nilsberth et al., 2001) and the deposition of amyloid
plaques. While β-secretase has been identified (for a review, see
Vassar and Citron, 2000), the nature of γ-secretase is still unclear
(De Strooper and Annaert, 2001). The two homologous presenilin
(PS) proteins, PS1 and PS2, which are critically required for the
intramembranous γ-secretase cut, may be aspartyl proteases
with γ-secretase activity (for a review, see Esler and Wolfe,
2001). This is supported by the identification of critical aspar-
tates within the putative transmembrane domains 6 and 7 of PSs
(Wolfe et al., 1999c), which may be part of a catalytically active
center of an intrinsic aspartyl protease activity (Wolfe et al.,
1999a). Moreover, γ-secretase inhibitors, which block Aβ gener-
ation, have been found to bind to PS1 (Esler et al., 2000; Li et al.,
2000b) and PSs share considerable homology around the critical
aspartate in transmembrane domain 7 with bacterial aspartyl
proteases (Steiner et al., 2000). However, additional co-factors
are required to allow formation of the biologically active PS
complex (Capell et al., 1998; Li et al., 2000a), and the cellular
distribution of PS does not necessarily reflect the location of
γ-secretase activity (Cupers et al., 2001). Thus, it is currently
unclear whether PSs are identical to the γ-secretase or just
support its targeting to its cellular sites of action (Cupers et al.,
PSs are not only essential for the γ-secretase cut of the Aβ
domain, but also for the highly similar site 3 (S3) cleavage of
Notch (De Strooper et al., 1999; for a review, see Mumm and
+Corresponding authors. Tel: +49 89 5996 472; Fax: +49 89 5996 415; E-mail: email@example.com or firstname.lastname@example.org
A. Geling and H. Steiner contributed equally to this work
EMBO reports vol. 3 | no. 7 | 2002 689
A γ-secretase inhibitor blocks Notch signaling in vivo
Kopan, 2000). This cut produces the Notch intracellular domain
(NICD), which translocates to the nucleus to regulate target gene
transcription (Mumm and Kopan, 2000). Ablation of the PS1 and
PS2 genes therefore results in a phenotype indistinguishable
from that caused by a Notch knockout (Donoviel et al., 1999),
and totally blocks Aβ and NICD production (Herreman et al.,
2000; Zhang et al., 2000). Moreover, mutagenesis of the critical
aspartates also blocks the function of human PS in Notch
signaling (Steiner et al., 1999). Therefore, inhibition of PS
activity not only blocks Aβ production, but also interferes with
NICD generation and the Notch pathway. Indeed, pharma-
cological inhibition of PS1 activity blocks Notch signaling in
cultured cells (De Strooper et al., 1999; Berezovska et al., 2000;
Martys-Zage et al., 2000; Doerfler et al., 2001; Hadland et al.,
2001). From a therapeutic point of view, this suggests that drugs
developed to lower Aβ production by interfering with γ-secretase
activity might affect PS function and therefore also block Notch
signaling in vertebrates. To prove whether a known γ-secretase
inhibitor (DAPT; Dovey et al., 2001) produces phenotypic side-
effects in a living vertebrate, we used zebrafish (Danio rerio) as
a model system. Our data not only demonstrate that a γ-secretase
inhibitor fully blocks Notch signaling in a living vertebrate, but
also suggest zebrafish as a suitable system to evaluate the effects
of Aβ-lowering drugs on Notch signaling in vivo.
As a prototype γ-secretase inhibitor, we investigated the highly
specific γ-secretase inhibitor DAPT (Dovey et al., 2001) for its
capacity to block Notch endoproteolysis. With this aim, HEK293
cells expressing endogenous PSs were stably transfected with
the Notch∆E cDNA [encoding a tagged version of the trans-
membrane and intracellular domains of Notch (Schroeter et al.,
1998)] and treated with or without DAPT. As shown in
Figure 1A, the NICD fragment is readily visible in untreated
cells, but its generation is inhibited by DAPT treatment. Next,
before testing the effects of DAPT in the zebrafish in vivo, we
verified that DAPT was also active on a zebrafish PS1 (zfPS1)-
controlled γ-secretase activity. We stably transfected HEK293
cells expressing Swedish mutant APP (HEK293/sw) (Citron et al.,
1992) with cDNA encoding wild-type (wt) zfPS1 or the non-
functional zfPS1 D374N mutant (Leimer et al., 1999). As
expected (Leimer et al., 1999), zfPS1 was endoproteolytically
processed, while endoproteolysis of zfPS1 D374N was blocked
and the full-length protein accumulated (Figure 1B). Endogenous
human PS1 and PS2 were replaced (Thinakaran et al., 1997) by
wt and mutant zfPS1, demonstrating that zfPS1 is incorporated
into the PS complex, the formation of which is required for
γ-secretase activity (Li et al., 2000a) (Figure 1B). Expression of
zfPS1 D374N caused a dramatic accumulation of the substrates
of γ-secretase, the APP C-terminal fragments (CTFs), which was
accompanied by an almost complete inhibition of total Aβ
(Aβ40 and Aβ42) generation (Figure 1B). In contrast, expression
of wt zfPS1 did not cause APP CTF accumulation and allowed
normal total Aβ production (Figure 1B). We next analyzed
Notch endoproteolysis in HEK293/sw cells stably co-expressing
Notch∆E and wt or D374N mutant zfPS1. Expression of wt zfPS1
allowed robust NICD production, which was strongly inhibited
by the zfPS1 D374N mutant (Figure 1C). Taken together, these
results demonstrate that zfPS1 is capable of controlling γ-secretase
and S3 protease activity in human cells. We therefore next inves-
tigated the effects of DAPT on APP and Notch endoproteolysis in
the presence of zfPS1-controlled γ-secretase and S3 protease
activity. DAPT caused a strong accumulation of APP CTFs with
concomitant inhibition of Aβ generation (Figure 1D). DAPT also
inhibited S3 cleavage of Notch in the presence of zfPS1. As
shown in Figure 1E, NICD production was inhibited by DAPT in
a dose-dependent manner. Thus, these experiments demonstrate
that DAPT efficiently blocks a zfPS1-dependent γ-secretase and
S3 protease activity.
We subsequently used the zebrafish system to assess the effect
of DAPT on Notch signaling at the morphological, molecular
and biochemical levels in vivo. A number of zebrafish mutants
Fig. 1. DAPT blocks Notch endoproteolysis and inhibits a zfPS1-dependent
γ-secretase and S3 protease activity in HEK293 cells. (A) HEK293 cells
stably transfected with the Notch∆E cDNA (Schroeter et al., 1998) were
treated with or without 1 µM DAPT for 4 h. Cell lysates were analyzed for
Notch∆E (N∆E; uncleavedform of Notch) and NICD (cleaved formof Notch)
by immunoblotting using antibody 9E10 to the myc tag at the C-terminus of
these Notch variants. (B) Panels 1–3: cell lysates from HEK293/sw cells
stably transfected with the indicated zfPS1 cDNAs were analyzed by
immunoblotting with antibody zfPS1loop, 3027 to human PS1 or 3711 to
human PS2. Note that endogenous human PS1 and PS2 are replaced by zfPS1
variants. Replacement of endogenous PSs by overexpressed PS variants is an
important indication for incorporation of the ectopic PS into a biologically
functional PS complex (Thinakaran et al., 1997). Panel 4: cell lysates were
analyzed for APP CTFs by immunoblotting with antibody 6687. Panel 5:
conditioned media were analyzed for total Aβ (Aβ40 and Aβ42) species by
immunoprecipitation/immunoblotting with antibodies 3926/6E10. Immuno-
precipitates were separated on a Tris–bicine–urea gel that allows the
identification of Aβ40 and Aβ42 (Wiltfang et al., 1997). Note that expression
of wt zfPS1 not only allows robust Aβ (Aβ40 and Aβ42) production, but also
leads to increased production of Aβ42 (Leimer et al., 1999). (C) HEK293/sw
cells expressing either wt zfPS1 or mutant zfPS1 D374N were stably
transfected with the Notch∆E cDNA (Schroeter et al., 1998) and cell lysates
were analyzed for Notch∆E (N∆E) and NICD as in (A). (D) HEK293/sw cells
stably expressing wt zfPS1 were treated with or without 1 µM DAPT for 4 h
and analyzed for APP CTFs as in (B) and for Aβ by direct immunoblotting
with antibody 3926. (E) HEK293/sw cells stably expressing wt zfPS1 were
treated with increasing amounts of DAPT for 4 h and cell lysates were
analyzed for Notch∆E (N∆E) and NICD as in (A).
690 EMBO reports vol. 3 | no. 7 | 2002
A. Geling et al.
affecting Notch signaling have been identified and display
characteristic early phenotypes, such as impaired segmentation
of the somites (van Eeden et al., 1996), and exacerbated primary
neurogenesis (Jiang et al., 1996). Their phenotypes constitute an
experimental counterpart with which the in vivo effects of DAPT
can be compared.
Treating zebrafish embryos with DAPT during the first 24 h of
development did not trigger gross morphological abnormalities,
but reproducibly impaired somite formation (91%, n = 54). At 24
h, somitic boundaries were misshapen, and delimited somites of
irregular size (Figure 2A and B). Morphological observations at
earlier stages indicated that, upon DAPT treatment, the first 4–8
somites formed normally while most posterior somites did not
(data not shown). This phenotype strikingly resembles zebrafish
Notch pathway mutants such as beamter (bea), deadly-seven
(des), after-eight (aei) and white-tail (wit) (Jiang et al., 1996;
van Eeden et al., 1996). At the molecular level, Notch activity
normally controls somite anteroposterior (AP) polarity, as well as
the cycling expression of somite prepatterning genes in the
presomitic mesoderm (Pourquie, 2000). To confirm our morpho-
logical analysis, we assessed the effect of DAPT on these two
Notch-dependent processes. Probing DAPT-treated embryos for
fgf8 and myoD expression, which respectively label the anterior
and posterior halves of each presumptive somite in the unseg-
mented mesoderm (Figure 2C and E), confirmed that DAPT, like
Notch deficiencies, affected somite AP polarity: fgf8 expression
was abolished upon DAPT treatment, while myoD expression
became ubiquitous (100%, n = 18 and 23) (Figure 2D and F).
The latter phenotype is similar to that reported in bea, des, aei
and wit mutants [see figure 7 in van Eeden et al. (1996), figure 5
in Jiang et al. (1996) and figure 5 in Holley et al. (2000)]. In the
region of nascent somites, Notch-dependent synchronized gene
cycling normally lays down a banded pattern of paraxial proto-
cadherin (papc) expression, which highlights the cells most
recently arrested in a somitic state (Yamamoto et al., 1998; Jiang
et al., 2000) (Figure 2G). papc expression is modified to a
randomized pattern in Notch signaling mutants [figure 3 in Jiang
et al. (2000)]. Similarly, we observed a random mixture of
papc-positive and -negative cells in the anterior presomitic
mesoderm of DAPT-treated embryos (Figure 2H) (100%, n = 12).
Thus, DAPT treatment affects somitogenesis in vivo in a manner
similar to deficiencies in Notch signaling.
We subsequently tested whether DAPT treatment also mimics
Notch signaling impairments during neurogenesis. Primary
neurogenesis in lower vertebrates involves the selection of
individual neuroblasts from proneural clusters by a Notch-
dependent lateral inhibition process (Lewis, 1998; Chitnis,
1999; Blader and Strahle, 2000). Specifically, analysis of
zebrafish mutants (Jiang et al., 1996) and in vivo misexpression
experiments (Dornseifer et al., 1997; Haddon et al., 1998; Takke
Fig. 2. DAPT affects somitogenesis (A–H) and neurogenesis (I–L) in vivo in a manner indistinguishable from Notch signaling deficiencies. Mock-treated (A, C,
E, G, I, K) and DAPT-treated (B, D, F, H, J, L) embryos visualized at 24 h [(A) and (B), live views] or at 15 somites (C–L), flat mounts following whole-mount
in situ hybridization with the probes indicated, anterior to the left and dorsal up except in (E) and (F) (dorsal views). (A–H), (K) and (L) are close-up views of the
trunk and tail; (I) and (J) are close-up views of the brain. DAPT treatment alters somitic borders [arrows in (A) and (B)]. It affects AP polarization of the somitic
mesoderm, normally revealed by the complementary expression of fgf8 and myoD [arrows in (C) and (E)], and randomizes expression of cycling-dependent genes
such as papc in nascent somites [arrows in (G) and (H)]. In the embryonic nervous system, DAPT triggers a neurogenic phenotype, with an increased number of
ngn1-positive neuroblasts in every proneural cluster [compare the clusters identified by arrows in (I) and (J), (K) and (L)]. D, presumptive diencephalon; ep,
epiphyseal cluster; i, intermediate neurons; m, motoneurons; s, sensory neurons; T, presumptive telencephalon; tel, telencephalic cluster; vrc, ventro-rostral cluster.
EMBO reports vol. 3 | no. 7 | 2002 691
A γ-secretase inhibitor blocks Notch signaling in vivo
et al., 1999) demonstrated that Notch signaling maintains its
expressing cells in an undifferentiated state, while neighboring
Delta-positive cells express the neuronal specification factor
neurogenin (Ngn1) (Blader et al., 1997) and generate neuro-
blasts. We observed that DAPT treatment strongly and reproducibly
increased the number of ngn1-positive cells within each
proneural cluster at all levels of the body axis during primary
neurogenesis (Figure 3I–L) (100%, n = 13), triggering a neuro-
genic phenotype similar to zebrafish mutants deficient in Notch
signaling, such as wit (Jiang et al., 1996), and to zebrafish
embryos rendered insensitive to Notch by misexpression of the
extracellular form of Delta (Haddon et al., 1998). Thus, like an
absence of Notch signaling, DAPT prevents the lateral inhibition
process of neuroblast selection in vivo.
The neurogenic phenotype resulting from Notch signaling
deficiencies can be reverted in vivo by overproduction of the
NICD fragment (see Haddon et al., 1998; Takke et al., 1999). To
further confirm that the neurogenic phenotype triggered by
DAPT in vivo resulted from impaired Notch processing, we
therefore tested whether it could be reverted by injection of nic,
an mRNA encoding the NICD fragment of zebrafish Notch1
(Takke et al., 1999). As concluded previously (Haddon et al.,
1998; Takke et al., 1999), local misexpression of NICD reduced
the number of ngn1-positive primary neurons in the embryonic
neural plate (Figure 3A and A′). This effect is epistatic to the
action of DAPT, as the number of primary neurons in
nic-injected areas was similarly reduced in DAPT-treated and
control embryos (compare Figure 3B and B′ and A and A′). Else-
where in the neural plate, and as documented above (Figure 2I–L),
neurogenesis was prominently enhanced by DAPT (Figure 3B
and B′). Thus, the neurogenic effect of DAPT can be reverted by
NICD, confirming that DAPT acts by interfering with Notch
signaling and upstream of NICD activity. Taken together, our
results demonstrate that DAPT affects zebrafish embryonic
development in a manner indistinguishable from Notch signaling
deficiencies, both at the morphological and molecular levels.
Our results demonstrate that DAPT, a known and carefully char-
acterized γ-secretase inhibitor (Dovey et al., 2001; Sastre et al.,
2001), severely interferes with Notch signaling in zebrafish
embryos. DAPT and other γ-secretase inhibitors were developed
as Aβ-lowering drugs thought to be used for long-term treatment
in human patients (Wolfe et al., 1999b; Dovey et al., 2001).
However, concerns about such a strategy were raised because it
could apparently interfere with the biological function of PS.
Based on numerous previous findings, PS clearly plays a role in
Notch signaling by facilitating NICD generation (for a review,
see Mumm and Kopan, 2000; Steiner and Haass, 2000). One
may therefore expect that such inhibitors not only have benefi-
cial effects with regard to Aβ production, but also unwanted
side-effects on the control of cellular differentiation via interfer-
ence with the Notch signaling pathway. Along this line, Hadland
et al. (2001) recently demonstrated that a distinct γ-secretase
inhibitor (Cpd.11) (Wolfe et al., 1999b) added to fetal organ
cultures represses thymocyte development, probably by
reducing Notch signaling. However, in this study, direct
evidence that proteolytic generation of NICD generation was
indeed affected in the CD4–/CD8– precursor cells was lacking.
Fig. 3. Expression of the soluble cytoplasmic domain of Notch prevents DAPT-mediated inhibition of Notch signaling. Flat-mounted, mock-treated (A and A′) and
DAPT-treated (B and B′) embryos visualized at 10 somites following in situ hybridization for ngn1expression (blue staining) (dorsal view, anterior to the left). All
embryos were injected into one blastomere at 4 cells with the NICD-encoding nic RNA and nlslacZ RNA as lineage tracer (brown nuclei identify the progeny of
the injected blastomere). (A′) and (B′) (same magnification) are enlarged views of the boxed areas in (A) and (B), respectively. Injected (+ nic) and non-injected
(uninj.) territories are indicated on either side of the embryo midline and boxed with a dotted line in (A′) and (B′). DAPT strongly increases the number of primary
neurons within the neural plate (Figure 2L and non-injected territory in Figure 3B′ compared with 3A′), while nic has the opposite effect (compare injected and
non-injected territories in A′). Note that NICD activity overrides the neurogenic effect of DAPT, as a similarly reduced number of neurons follows nic expression
with or without DAPT [compare injected sides in (A′) and (B′)].
692 EMBO reports vol. 3 | no. 7 | 2002
A. Geling et al.
We now demonstrate for the first time that a γ-secretase inhibitor
interferes with Notch signaling in vertebrates in vivo, directly
suggesting that rather significant putative side-effects are to be
expected from such drugs during long-term treatment in
The detrimental effects of DAPT were observed during embryo-
genesis of zebrafish. However, they are likely to occur in adults
as well, as Notch signaling is active at all stages and in multiple
tissues. For example, hematopoiesis is required throughout life
and thymocyte differentiation requires Notch signaling (Hadland
et al., 2001). All Notch factors are also expressed in the adult
brain (Weinmaster et al., 1992; Higuchi et al., 1995; Berezovska
et al., 1998; Irvin et al., 2001), where they are likely to play
pivotal roles in terminally differentiated neurons, as well as in
the control of gliogenesis and neural stem cell differentiation.
However, a conditional knockout of the PS1 gene had no
obvious effects on Notch signaling in mice (Yu et al., 2001). The
lack of effects on Notch signaling is likely to be due to the
abundant expression of PS2 (Yu et al., 2001), which supports
Notch signaling like PS1 (Steiner et al., 1999). In contrast to the
conditional PS1 depletion, γ-secretase inhibitors will affect both
PS1 and PS2 function (Esler et al., 2000; Li et al., 2000b). Besides
putative side-effects on Notch signaling, PSs bind β-catenin, thus
independently interacting with yet another signaling pathway
potentially controlling cell proliferation in adults. Indeed, loss of
PS1 in mice also results in enhanced β-catenin signaling, which
causes skin tumors in adult mice (Xia et al., 2001).
In summary, our data not only demonstrate that a γ-secretase
inhibitor blocks Notch signaling in a living vertebrate, but also
provide a novel model system for the validation of drugs that
differentially affect Aβ production and NICD formation (Petit
et al., 2001). After only 24 h, numerous zebrafish embryos can
be investigated for deficits in somitogenesis or neurogenesis,
which provide a precise and reliable read-out of Notch signaling
activity. Therefore, our results identify the zebrafish as a valuable
test system for the validation of Aβ-lowering drugs that do not
interfere with other physiologically important signaling pathways.
Cell lines and cell culture. HEK293 cell lines were generated
and cultured as described previously (Steiner et al., 2000).
Antibodies. The polyclonal antibodies against PS1 (3027) and
PS2 (3711), against zfPS1 (zfP1loop) (Leimer et al., 1999), against
the C-terminus of APP (6687) and against Aβ1–42 (3926) have
been described previously (Steiner et al., 2000). Monoclonal
antibodies against Aβ1–17 (6E10) and the c-myc epitope (9E10)
were obtained from Senetek (6E10) and from the Developmental
Studies Hybridoma Bank, University of Iowa (9E10).
PS, APP and Notch endoproteolysis. Analysis of human and
zebrafish PS expression was as described previously (Leimer
et al., 1999; Steiner et al., 2000). APP CTFs were analyzed as
described before (Steiner et al., 2000) and Aβ production was
analyzed by combined immunoprecipitation/immunoblotting of
conditioned media with antibodies 3926/6E10 as described
previously (Sastre et al., 2001) or by direct immunoblotting of
aliquots of conditioned media with antibody 6E10. Notch endo-
proteolysis was analyzed by immunoblotting of cell lysates with
Fish strains. Embryos were obtained from natural spawning of
wild-type (AB strain) adults; they were raised and staged
according to Kimmel et al. (1995).
DAPT treatments. A 10 mM stock of DAPT in DMSO was
diluted in embryo medium and applied to dechorionated
zebrafish embryos at 28°C from the sphere stage (late blastula)
until the stage of analysis (see figure legends). Control embryos
were mock treated with embryo medium containing the same
concentration of DMSO carrier only. We first performed a dose–
response analysis and established that a minimal concentration
of 50 µM DAPT was required to affect somitogenesis at the
morphological and molecular levels (30 and 60% of cases,
respectively, n = 34; not shown). All the results reported here
were obtained using a dose of 100 µM DAPT. HEK293 cells
were treated with 1 µM DAPT for 4 h.
Capped mRNA injections in zebrafish embryos. nic capped
RNA (encoding the NICD of zebrafish Notch1) was synthesized
as described by Takke et al. (1999) using the mMessage
mMachine kit (Ambion), and 5 pg were injected (together with
4 pg of nlslacZ RNA as lineage tracer) into a single blastomere
of 4-celled embryos. Nucleus-localized β-galactosidase was
revealed using rabbit anti-β-galactosidase (1/4000) followed by
goat anti-rabbit–HRP (1/200; Jackson Laboratories) antibodies
and DAB revelation.
In situ hybridizations and immunocytochemistry. In situ
hybridizations were carried out according to standard protocols
(Thisse et al., 1993) using the following probes: fgf8 (Reifers et al.,
1998), myoD (Weinberg et al., 1996), papc (Yamamoto et al.,
1998) and ngn1 (Blader et al., 1997).
We thank the Boehringer Ingelheim Pharma KG for the gift of
DAPT and S. Amacher, P. Blader, J.A. Campos-Ortega,
D. Edbauer, R. Kopan, U. Leimer, C. Thisse and E. Weinberg for
providing cDNAs, antibodies and cell lines, and G. Basset for
technical assistance. We thank R. Baumeister, C. Goridis and
W. Wurst for critically reading this manuscript, and H. Takeda
for communicating unpublished data. This work was supported
by grants from the Deutsche Forschungsgemeinschaft (to C.H.
and H.S.), the European Community (to C.H.) and the Volkswagen-
Stiftung (to A.G. and L.B.-C.).
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