Apaf1 (CED-4 Homolog) Regulates Programmed Cell Death in Mammalian Development

Abstract

The cytosolic protein APAF1, human homolog of C. elegans CED-4, participates in the CASPASE 9 (CASP9)-dependent activation of CASP3 in the general apoptotic pathway. We have generated by gene trap a null allele of the murine Apaf1. Homozygous mutants die at embryonic day 16.5. Their phenotype includes severe craniofacial malformations, brain overgrowth, persistence of the interdigital webs, and dramatic alterations of the lens and retina. Homozygous embryonic fibroblasts exhibit reduced response to various apoptotic stimuli. In situ immunodetection shows that the absence of Apaf1 protein prevents the activation of Casp3 in vivo. In agreement with the reported function of CED-4 in C. elegans, this phenotype can be correlated with a defect of apoptosis. Our findings suggest that Apaf1 is essential for Casp3 activation in embryonic brain and is a key regulator of developmental programmed cell death in mammals.

Full text

Cell, Vol. 94, 727–737, September 18, 1998, Copyright 1998 by Cell Press
Apaf1 (CED-4 Homolog) Regulates Programmed
Cell Death in Mammalian Development
proteases termed caspases (cysteine aspartate prote-
ases). These proteases initiate the apoptotic proteo-
lytic cascade that leads, for instance, to activation of
Francesco Cecconi,* Gonzalo Alvarez-Bolado,*
Barbara I. Meyer,* Kevin A. Roth,
†
and Peter Gruss*
‡
nucleases and cleavage of nuclear structural proteins*Department of Molecular Cell Biology
(reviewed in Takahashi and Earnshaw, 1996).
Max Planck Institute of Biophysical Chemistry
Recently, a human CED-4 homologhas been isolated
D-37077 Go
¨
ttingen
in HeLa cells and termed APAF1 (apoptotic protease
Germany
activating factor1; Zou et al., 1997). APAF1 participates
†
Department of Pathology
in the cytochrome c/dATP-dependent activation of a
Washington University School of Medicine
mammalian CED-3 homolog, CASPASE 3(CASP3) (also
St. Louis, Missouri 63110
namedCPP32, Xueetal.,1996;Zou etal.,1997),through
the proteolytic activation of CASP9 (another CED-3 ho-
molog; Duan et al., 1996; Li et al., 1997).
Summary
CED-4 has been determined to function downstream
of CED-9 but upstream of CED-3 (Shaham and Horvitz,
The cytosolic protein APAF1, human homolog of C. ele-
1996).CED-3,CED-4, andCED-9formaternarycomplex
gans CED-4, participates in the CASPASE 9 (CASP9)-
in C. elegans (Chinnaiyan et al., 1997; Spector et al.,
dependent activation of CASP3 in the general apo-
1997). Likewise, APAF1 functions downstream to Bcl2
ptotic pathway. We have generated bygene trap a null (or its close family member BclX
L
), which regulates the
release of cytochrome c from mitochondria, but up-alleleof themurine Apaf1. Homozygousmutantsdie at
stream of CASP3 (Zouetal., 1997). Besides, CASP9 andembryonic day 16.5. Their phenotype includes severe
BclX
L
bind distinct regions of APAF1 forming also incraniofacial malformations, brain overgrowth, persis-
mammalian cells a ternary complex (Pan et al., 1998).
tenceof the interdigitalwebs,anddramatic alterations
The evolutionary conservation among C. elegans and
of the lens and retina. Homozygous embryonic fibro-
vertebrates of the general apoptotic program at a bio-
blasts exhibit reduced response to various apoptotic
chemical andcellularlevel isthusevident,buttheimpor-
stimuli. In situ immunodetection shows that the ab-
tance of apoptosis in animal development grows with
sence of Apaf1 protein prevents the activation of Casp3
organism complexity. Apoptosis-deficient nematodes
in vivo. In agreement with the reported function of
can have a normal life span, although they have 15%
CED-4 in C. elegans, this phenotype can be correlated
more cells than normal animals and show a few func-
with a defect of apoptosis. Our findings suggest that
tional deficiencies (Ellis et al., 1991; Jacobson et al.,
Apaf1 is essential for Casp3 activation in embryonic
1997). By contrast, mice in which Casp3 has been de-
brain and is a key regulator of developmental pro-
leted by targeted disruption die perinatally with a mas-
grammed cell death in mammals.
sive cell overgrowth in the central nervous system, as
a result of apoptosis deficiency in the neuroepithelial
Introduction
cells (Kuida etal., 1996). BclX-deficient mice die earlier,
at embryonic day 13, exhibiting excess of apoptosis in
Apoptosis is an evolutionary conserved form of cell
brain, spinal cord, and hematopoietic system (Moto-
death regulated by several genes that play crucial roles
yama et al., 1995).
in the development and homeostasis of multicellular
Apoptosis servesthreefunctionsinmammaliandevel-
organisms (Kerr et al., 1972; Jacobson et al., 1997).
opment (Glu
¨
cksmann, 1951): deleting unneeded struc-
In the nematode C. elegans, several genes have been
tures (phylogenetic apoptosis), controlling cell number
isolated withnegativeand executive effects on the gen-
(histogenetic apoptosis), andsculpting structures (mor-
eral apoptotic program, among them, ced-9, which in-
phogenetic apoptosis). It remains to be determined
hibits cell death (Hengartner et al., 1992), and ced-3
when and to which extent each protein member of the
and ced-4, which promote cell death (Yuanand Horvitz,
apoptotic program is involved in each of these pro-
1990, 1992).
cesses. Obviously, Apaf1 is a good candidate to play a
Bcl2represents the mammalian counterpart ofCED-9
crucial role in mammalian development, for it interacts
(Vaux et al., 1992; Hengartner and Horvitz, 1994) and is
with BclX
L
and is upstream of Casp3.
an integral membrane protein located mainly on the
Toaddressthesequestions,we havetakenadvantage
outer mitochondrial membrane (Krajewski et al., 1993).
of the results of a gene trap study (Chowdhury et al.,
Bcl2 belongs to a multigene family with several repre-
1997) that has providedthemeans to isolatea null allele
sentatives in mammals (reviewed in Farrow and Brown,
of the murine Apaf1 gene. Here, we report the cloning
1996; Reed, 1997; Newton and Strasser, 1998). CED-3
of the mouse Apaf1 cDNAandthe analysisof embryonic
encodes a protein 29% identical to human interleukin
phenotype in mice homozygous for this mutation. Our
(IL)-1b-converting enzyme(ICE,Yuanetal., 1993),which
results indicate essential roles in vivo of Apaf1 in (1)
belongs as well to a large family of related mammalian
regulating Casp3 activation, (2) establishing the proper
development of brain structures, (3) controlling the cell
number of retinal cell populations and lens polarization
in eye development, (4) sculpting digits by eliminating
‡
To whom correspondence should be addressed.
Open access under CC BY-NC-ND license.

Cell
728
Figure 1. Generation of Apaf1-Deficient Em-
bryos and Sequence of Apaf1 Protein
(A) Structure of the gene trap-vector/Apaf1
mRNA fused transcript. CARD, caspase re-
cruitment domain; CED-4, CED-4-like do-
main;SA, spliceacceptor sitefrom En2gene;
IRES, internal ribosomal entry site of the en-
cephalomyocarditis virus; b-GEO, b-galactosi-
dasefused toneomycin phosphotransferase;
SVpA,SV40 polyadenylation site(Chowdhury
et al., 1997).
(B)Northernblotofpoly(A)1 RNAfrommouse
embryos at several developmental stages,
hybridized with the 59RACE product derived
from the Apaf1 gene trap line.
(C) Northern blot of poly(A)1 RNA from wild-
type, homozygous, and heterozygous e14.5
embryos. Black arrowheads point to the en-
dogenous (lower) and fusion (upper) tran-
scripts.
(D) Westernblotof proteinextractsfromwild-
type, homozygous, and heterozygous e11.5
embryonic brains. Open arrowhead points to
the endogenous Apaf1 protein band.
(E) Comparison of murine and human Apaf1
protein sequences.Gray box, CARD domain;
white box, CED-4-like domain; underlined
black, WD40 repeats; underlined gray (indi-
cated by a dot in [A]), an additional WD40
repeat present in the mouse Apaf1 protein and
deducedfrom thehumanKIAA0414 cDNAse-
quence (Ishikawa et al., unpublished data,
GenBank accession number AB007873). Ar-
rowhead, gene trapinsertion site fusion tran-
script.
interdigital cells, and (5) leading the processes of sec- internal ribosome entry site [IRES]), under the control
of the Apaf1 gene regulatory elements (Figure 1A).ondary palate formation. These observations imply the
possible involvement of Apaf1 in alternative cell death HumanAPAF1possessesanNH
2
-terminalCED-3pro-
domain–like region that includes a CARD (caspase re-regulatory pathways.
Involvement of cell death genes in many human dis- cruitment domain), a CED-4-like segment, a COOH-ter-
minal extension composed of multiple WD40 repeatseases has been shown in several cases (reviewed in
Hoeppner et al., 1996). The localization of the APAF1 thatarelackingin thenematode CED-4 (Zouet al.,1997;
Figure 1E). The insertion within the murine Apaf1 oc-gene on the long arm of human chromosome 12 sug-
gestsa roleforthis genein Noonansyndrome(Jamieson curred at the end of the region coding for the ninth
of these repeats. As shown in experiments of in vitroet al., 1994).
reconstitution, the wild-type CASP9 translated in vitro
binds to the CARD of APAF1 (Li et al., 1997). However,Results and Discussion
these data do not exclude the possibility that CASP9
interacts with other regions of APAF1 aswell. Likewise,Generation of a Null Allele of the Murine
Apaf1 Gene BclX
L
has been shown to bind a truncated APAF1 form
that contained only the CED-4 homologous region (PanIn a large-scale gene trap screening program, we have
isolated an embryonic stem (ES) cell clone trapping a etal., 1998).Therefore,CASP9 andBclX
L
bindto distinct
domains in APAF1, consistent with the formation of agene that showed aninteresting spatiotemporal expres-
sion pattern (see Figures 3–4). Using 59RACE from the ternary complex. Finally, Zou et al. (1997) showed that
APAF1 and CASP9 form a complex only in thepresenceknown reporter gene sequences, a 505-base-pair (bp)
long fragment of theendogenous gene wascloned. The ofcytochromecanddATP,whichmayinduceconforma-
tional changes in APAF1 that expose its CARD.sequenceof the59RACEproductrevealedafusionofthe
reporter bgeo gene(b-galactosidase fused toneomycin The WD40repeats at the COOH terminusare believed
to mediate protein–protein interactions (Neer et al.,phosphotransferase; Skarnes et al., 1992) to the murine
homolog of Apaf1 cDNA, 39 downstream of the position 1994). Recently, the APAF1 WD40 domain has been
shown to interact with BclX
L
, but the meaning of thisencoding the amino acid (aa) 1018 (corresponding to
number 975 of the 1194 aa composing the human pro- interaction is unknown (Hu et al., 1998). Moreover,dele-
tion of the WD40 repeats renders APAF1 constitutivelytein; Zou et al., 1997). Theinsertion predicts the expres-
sion of a truncated Apaf1 protein and of the reporter active and capable of processing CASP9 independent
of cytochrome c and dATP (Srinivasula et al., 1998).protein bgeo (independently translated by means of an

Apaf1 Regulates Apoptosis in Mouse Development
729
Table 1. Embryonic Lethality of Apaf1 Insertional Mutation
No. of Genotype of Embryos No. of
Total Normal-Looking Abnormal
Age (Days) No. of Litters Conceptuses Embryos 1/11/22/2Embryos
e10.5–11.0 1 12 12 5 5 2 0
e11.5–12.0 4 45 37 13 24 8 0
e12.5–13.0 2 19 14 4 10 5 5
e14.5–15.0 5 55 43 13 30 12 13
e16.5–17.0 4 43 39 11 28 4 4
e17.5–18.0 3 25 25 8 17 0 0
Total 19 199 170 54 114 31 22
Murine Apaf1 mRNA is expressed at high levels from Apaf1 Mutant Mice Develop Abnormally
and Die before Birth
at least e11.5 to e17.5, as revealed by Northern blot
We examined about 200 embryos of the Apaf1 geneanalysis, anditis about 7.0kb insize, in agreementwith
trap line (Table1).Abnormalembryos werefoundonly atthereported dimensionof its human counterpart (Figure
e12.5and later.Allabnormalembryoswerehomozygous1B). Southern analysis of genomic DNA allowed the ge-
for the Apaf1 mutation, with only one exception; one
notyping of an e14.5 litter (datanot shown). Subsequent
wild-type e14.5 embryo showed exencephaly. The het-
Northernblot analysiswasusedtoidentifyrelativelevels
erozygous animals were healthy and of normal size. No
of wild-type and fusion transcripts in wild-type, hetero-
homozygous embryos were found beyond e16.5, sug-
zygous, and homozygous embryos (Figure 1C). In the
gestingthat the Apaf1 mutationislethal aroundthis age.
homozygotes, no endogenous Apaf1 mRNA was de-
The relative proportions of homozygous, heterozygous,
tected,reflecting the absence of any splicing ofthepre-
andwild-type embryosfound inour studyareconsistent
mRNA transcribed from the mutant alleles around the
with the expected Mendelian ratio. The phenotype of
newly inserted exons.
e12.5, e14.5, and e16.5 mutant embryos was analyzed
Accordingto theinsertionalsite,themutant transcript
and compared to the phenotype of the corresponding
(8.9 kb: 3.6 kb of the endogenous mRNA fused to 5.3
wild-type littermates.
kb of the insertional vector mRNA) is predicted to code
Since no apparent discernible histologicalabnormali-
for two proteins; one of them would comprise (1) the
ties were observed in the developing heart, lung, and
CARDdomain, (2)the CED-4-likedomain, and (3) a trun-
liver (despite expression of Apaf1 in these tissues in
cated WD40 repeats domain of Apaf1; the second pro-
normal adult animals; data not shown), we focused on
tein would consist of bgeo as the reporter product. To
the craniofacial, eye, limb, and brain alterations.
determine whether a truncated Apaf1 protein was pres-
ent in the mutant homozygous embryos, we carried out
Apaf1 Mutant Embryos Show
immunoblotting experiments using antibodies raised to
Craniofacial Alterations
the NH
2
terminus of the humanAPAF1 protein. The anti-
Fetuses (e16.5) homozygous for the Apaf1 mutation
APAF1 antibody recognizes a protein that has an ap-
showacharacteristiccraniofacialphenotype whosema-
proximatemolecular weight of 135 kDa.Analysis ofpro-
jor traits are midline facial cleft, absence of skull vault,
tein extracts from an e12.5 litter is shown in Figure 1D.
and of all vomer and ethmoidal elements, rostral exen-
In theApaf1
2
/
2
embryos,there was no detectable Apaf1
cephaly, and cleft palate(Figures 2A–2H).Targeted mu-
protein, as compared with the wild-type littermates. It
tation of anumber of geneshas been reported to cause
is most likely that the truncated protein, if synthesized,
varying degrees of facial cleft, palatal cleft, or both (re-
is not stable and is rapidly degraded (Capecchi et al.,
viewed in Richman and Mitchell, 1996). None of these
1974; Rechsteiner, 1987; Subramanian et al., 1995).
genes, however, encodes acomponent of theapoptotic
machinery. Nevertheless, apoptosis has been hypothe-
Cloning and Sequence of Murine Apaf1 cDNA
sized to play a key role in palatal fusion processes,
We used the 59RACE product to probe a murine e15.5
where it would eliminate the medial edge epithelium of
cDNA bank in order to clone the wild-type Apaf1 mes-
the secondarypalatal shelvesafter theyhavecontacted
sage. The conceptual translation of the obtained 5227
at e14.5 in the midline (reviewed in Ferguson, 1988).
bp long Apaf1 cDNA sequence is shown in Figure 1E.
Accordingly, late and imperfect palatal fusion is a key
Using the basic alignment tool (BLAST, Altschul et al.,
component of the Apaf1 mutantphenotype (Figures2C–
1990), the entire length of the sequence was compared
2D). The basisphenoid bone, which is normally involved
to the most commonly used databases. The 1238 aa
in the formation of the caudal third of the palate, shows
protein is 88% homologous to the human APAF1 (Zou
an ossification defect in our mutants (Figures 2G–2H).
et al., 1997).
It is tempting to speculate that proper fusion in the
The gap of alignment between aa 811 and aa 855
midline of the palate is an essential step for basisphe-
among Apaf1 orthologs can be accounted for by the
noid bone formation.
presence of a supplementaryWD40 repeatin themouse
Our resultssuggest thatapoptosis is essential in mid-
sequence; the human KIA00414 partial mRNA, identical
line fusion of craniofacial structures. Since the Apaf1
to human APAF1, also contains this insertion (see Fig-
mutant phenotype is the only apoptosis-deficient phe-
notype showing midline fusion defect, Apaf1 could beure 1E).

Cell
730
part of aspecific apoptotic pathway involved in midline
fusion.
Persistence of Interdigital Webs in Apaf1
Mutant Embryos
In wild-type and heterozygous embryos, the interdigital
cells of the limbs, which undergo apoptosis as a means
of sculpting the digits in many vertebrates (Saunders,
1966; reviewed in Jacobson et al., 1997), disappeared
by e15.5; in the homozygotes at this stage, however,
these cells can still be seen (Figures 2I–2K). Apoptosis
wasdetected insituby DNA fragmentationlabeling(Fra-
gEL method) in the wild-typebut only ata verylow level
in the homozygous interdigital cells (data not shown).
These findings not only confirm in vivo the need for
programmed cell death in the sculpting of the normal
limb but also support the hypothesis that Apaf1 is a key
component in apoptosis in multiple cell types.
Abnormal Eye Development in Apaf1
Mutant Embryos
Apaf1 mutants show alterations of the retina, lens, and
eye vascular system. Already by e10.5 the retina shows
Apaf1 expression (Figure 3A), although at that age the
eyeprimordiumofthemutantismorphologically normal.
By e12.5, the retina of the mutant is noticeably thicker
than the retina of the wild-type littermate (Figures 3B
and 3C). At e14.5, the hyperplasic retina occupies most
of the optic cup and is folded (Figures 3D and 3E). Pro-
grammed cell death has been described as a regulator
of cell number (Bunt and Lund, 1981) during normal
development of the retina (histogenetic cell death). Re-
cently, it has been reported that two mechanisms of
apoptosis coexistin the developing retina; one of these
mechanisms is characteristic of retinal ganglion cells
(Rehen et al., 1996). The specific expression of Apaf1
in the outermost layer of the developing retina (corre-
spondingtothedeveloping ganglioncelllayer)suggests
that this factor could be part of a ganglion cell–specific
apoptotic pathway.
The lens of the Apaf1 mutant is smaller and seems
incorrectly polarized. Programmed cell death has been
reported to have a major role in lens morphogenesis
(Silver and Hughes, 1973). The correct polarization of
the lens depends probably on diffusible factors in the
Figure 2. Craniofacialand LimbPhenotypeoftheApaf1
2
/
2
Embryos
aqueous and vitreous (Coulombre and Coulombre,
(A and B) Facial midline cleft and rostral exencephaly in a homozy-
1963). It is conceivable that alterations in the size and
gous e16.5 fetus (A), as compared toa wild-type littermate (B). Red
shape of the lens primordium subsequent to defective
arrowheads, rostral border of the whisker pad. FB, forebrain; ton,
morphogenetic apoptosis lead to incorrect interaction
tongue.
(C) Transversal section through the caudal third of the palate of an
with the environment and thus to incorrect polarization.
e14.5 homozygous embryo. The palatal shelves meet in the midline
Apaf1is expressed inthe endothelial cellsof the tran-
(arrowhead) but do not fuse. pal, secondary palate.
sient vascular system of the eye (hyaloid capillary sys-
(D) Transversal section through the caudal third of the palate of an
tem).By e14.5 vascular endothelialcellsseem toobliter-
e14.5 wild-type embryo, showing complete fusion of the palatal
atecompletelytheoptic cup of the Apaf1mutant (Figure
shelves in the midline (arrowhead). pal, secondary palate.
(E) Composite figure comparing transverse sections through the
rostral facialstructures of a mutant e16.5 fetus(left) and a wild-type
littermate (right).In themutant, thebrain separates thenasalcavities
view from top. There is a large defect in the rostral two-thirds of the
(arrowheads), while the tongue and jawhave a normal appearance.
palate (asterisk), and the basisphenoid ossification point is absent.
MB, midbrain; OB, olfactory bulb; ton, tongue.
(H) Wild-type littermate. Theskull vault has beenremoved. bo,basi-
(F) Composite figure comparing transverse sections through the
occipital; bs, basisphenoid; lat. max., lateral maxillary bone; pt,
caudal facial structures of a mutant e16.5 fetus (left) and a wild-
pterygoid bone.
type littermate (right). In the mutant, the skull is defective and the
(I, J, and K) Comparison of the right hands of e15.5 mouse fetuses
brain is grossly mispositioned. FB, forebrain; HB, hindbrain.
reacted for detection of lacZ activity. The interdigital webs are per-
(G) Skeletal preparation of the skull of an e15.5 homozygous fetus;
sistent in the homozygous (arrowhead).

Apaf1 Regulates Apoptosis in Mouse Development
731
In summary,theApaf1phenotype underlinesthethree
different functions of apoptosis in the eye, cell number
regulation (histogenetic cell death, in the retina), mor-
phogenetic cell death (lens), and elimination of a tran-
sient structure (phylogenetic cell death, in the hyaloid
capillary system).
Brain Hyperplasia in Apaf1 Mutant Embryos
From e12.5 onward, the brain of the Apaf1 mutants
shows important morphological distortion. The telence-
phalic vesicles seem abnormally folded and reduced in
size, presumably due to the pressure exerted by the
overgrown diencephalon and midbrain (Figures 4A and
4B). Anatomically, the brain hyperplasia found in our
mutants is particularly intense in the diencephalon and
midbrain; the convoluted mass of neuroepithelium and
mantle that occupies the lumen of the midbrain (Figure
4C) is an abnormally enlarged choroid plexus of the
fourth ventricle (hindbrain;Figure4D). Histologically,the
hyperplasia is localized to the mantle layer (differentiat-
ing compartment), especially in the diencephalon (Fig-
ure 4C). The ventricular layer (mitotic compartment)
seems affected only in the choroidplexus. In this struc-
ture, not only the neuroepithelium is extensively over-
grownandfolded,butitisableto generateamantlelayer
(Figure 4D), absent in wild-type animals. The medulla
(hindbrain) is of normal appearance, and it is possible
to identify nuclear primordia (Figure 4E). The cortical
primordium is apparently delayed in development, but
the size and organization of the ventricular layer seem
normal (Figures4F and 4G). The onsetofthis phenotype
is an overgrowthof the ventralside of thehypothalamus
insinuating itself throughthe cartilage ofthe base ofthe
skull ate12.5.Thisphenomenonismost evidentat e14.5
(Figure 4H). Consistently with this early defect, Apaf1 is
expressed early in the ventral diencephalon (Figures 4I
and4J).ExpressionofApaf1asrevealedby lacZstaining
Figure 3. Eye Alterations in Apaf1 Mutants
can alsobe seenin the earlymantle of thebasal ganglia
(A) Vibratome section through the eye of an e10.5 heterozygous
(Figures4I and4K), intheneuroepitheliumof the choroid
mouseembryo reactedforlacZ activitydetection. Arrowhead,local-
ized expressionof Apaf1 in theneural retina.Rne, neural retina;Rpi,
plexus of the fourth ventricle (Figure 4L), and in the
pigmented retina.
marginal layer of the hindbrain (Figures 4I and 4M) and
(BandC)The eyeofthee12.5homozygousembryoshowsthickened
spinal cord.Thecauseofthebrainphenotypeis presum-
retina and small lens (B) as compared to the eye of a heterozygous
ably the lack of normal developmentalprogrammed cell
littermate (C). Embryos were reacted for lacZ activity detection be-
death in the mantle layer (differentiating compartment)
foresectioning,and sectionswere counterstainedwith hematoxylin-
of the diencephalon, midbrain, and cerebellum as well
eosin. Both homozygous and heterozygousshow Apaf1 expression
in the ganglion cell layer of the retina (yellow arrowheads) and in
as in the ventricular layer (mitotic compartment) of the
the endothelial cells of the vascular system (red arrowheads). Rne,
choroid plexus of the fourth ventricle. In wild-type ani-
neural retina; Rpi, pigmented retina.
mals, the choroid plexus is a region of neuroepithelium
(D and E) Sections throughthe eyes of homozygous (D) and hetero-
that does not generate a mantle layer but differentiates
zygous (E) e14.5 embryos. The homozygous retina is thicker, and
into an epithelial monolayer. Apoptosis is known to be
the lensis smaller. Endothelial cells fill the optic cupin the homozy-
a major normal developmental mechanism in the brain;
gous. The portion framed in (D) is shown enlarged in the inset.
Redarrowheads, endothelial cells;yellow arrowheads, ganglion cell
in this organ, programmed cell death has mostly histo-
layer. Rne, neural retina; Rpi, pigmented retina.
genetic functions (reviewed in Jacobson, 1991). The
phenotypes caused by the targeted mutation of either
of two other major genes related to programmed cell
3D). This phenomenon suggests that apoptosis is nec-
death agree with this view. Mouse embryos deficient in
essary not only postnatally (reviewed in Lang, 1997) to
the Casp3 gene show alterations in brain developmenteliminate the transient hyaloid capillary system but also
due to lack ofnormal amount of programmed cell death
to regulate the number of hyaloid capillaries already in
(Kuida et al.,1996). Deficiency in BclX
L
, anantiapoptotic
the prenatal period. Alternatively, it is possible that the
protein, has as a result abnormally extensive death of
reduction in the size of the optic cup of the mutants
neurons (Motoyama et al., 1995). In both cases the cell(consequent to the abnormal increase in retinal thick-
population affected by the mutation is the differentiat-
ness) is the cause of a concentration of the hyaloid
capillaries in the center of the eye. ing, postmitotic neurons of the mantle layer. The Apaf1

Cell
732
mutants, however, show both an excess of differentiat-
ing neurons in the mantle layer of midbrain and dien-
cephalon andan abnormally large mitotic layer (ventric-
ular layer) in the choroid plexus of the fourth ventricle.
This suggests that Apaf1 has a role in morphogenetic
as well as histogenetic programmed cell death in the
developing nervous system.
The obliteration of the lumen of the neural tube could
have as a consequence a degree of hydrocephalia. The
enlarged brain, compoundedby thedefects of skulland
the facial midline cleft and by the hydrocephalia, has
as a final result the gross exencephaly found in Apaf1
mutants by e16.5 (Figures 2A and 2E).
Apaf1 Is Required for Activation
of Casp3 In Vivo
The similarity of brain phenotypes in Apaf1 and Casp3
null mutations (Kuida et al., 1996; this work) strongly
indicates that Apaf1 and Casp3 can be components of
the sameapoptotic pathway during brain development;
this would be consistent with their functional interac-
tions, already shown in vitro (Li et al., 1997; Zou et al.,
1997). We have confirmed this hypothesis by means of
immunostaining experiments carried out with the CM1
antibody on histological sections of wild-type and
Apaf1
2
/
2
e12.5 embryos. The CM1 antibody, recently
characterized (Srinivasan et al., 1998), recognizes only
the cleaved 17 kDa subunit but not the 32 kDa proen-
zyme of Casp3. In the the wild-type central nervous
system, we observed numerous CM1-immunoreactive
cells (Figures 5A and 5B, 5E and 5F); positive neuritic
processes were also readily identifiable (arrows in Fig-
ures 5E and 5F). In contrast, little if any CM1 immunore-
activity was detected in the e12.5 Apaf1 homozygous
brain (Figures 5C and 5D, 5G and 5H). Control studies
performedon wild-typeembryosshowedno immunore-
activity in sections incubated without primary antibody.
CM1 has previously been used to demonstrate Casp3
arecomparable, althoughthe homozygous seemsdelayed in devel-
opment. ven, ventricular layer; il, intermediate layer; wt, wild type.
(H) Nissl-stained transverse sectionshowing the ventral diencepha-
lon of an e14.5 homozygous embryo. There is an abnormal growth
Figure 4. Brain Phenotype of Apaf1 Mutants
of nervous tissue (arrowhead) insinuating itself through the base of
(A) Nissl-stainedtransversesection throughtheforebrain ofane12.5 the skull (asterisks).
homozygous mouse embryo showing folding of the cortex (red ar- (I) Transverse vibrotome section through the forebrain of an e10.5
rowhead) anddeformation of theganglioniceminence(red asterisk). heterozygous mouse embryo reacted for lacZ activity detection.
The optic sulcus (yellow arrowhead)and trigeminal ganglion (yellow The incipient mantle layers of the basal ganglia and ventral dien-
asterisk) are landmarks for comparison with (B). (B) Nissl-stained
cephalon, aswell as the marginal layer of the hindbrain, are labeled
transverse section through the forebrain of a wild-type e12.5 em-
(arrowheads). FB, forebrain; HB, hindbrain; sopt, optic sulcus.
bryo. Asterisks and arrowheads as in (A).
(J) Detail of (I) showing Apaf1 expression in the mantle layer of the
(C) Hematoxylin-eosin-stained transverse sectionthrough the dien-
ventral diencephalon (arrowhead). INF, infundibulum; mtl, mantle
cephalon and rostral midbrain of an e14.5 homozygous embryo.
layer; RP, Rathke’s pouch; ven, ventricular layer.
The mantle layer (mtl) and choroid plexus (chp) show overgrowth
(K) Detail of (I) showing Apaf1 expression in the mantle layer of
and distorted shape. Ectopic masses of cells occupy the lumen
the pallidal ridge (primordium of basal ganglia; arrowhead). FBV,
(asterisk).
forebrainventricle;INF, infundibulum;mtl,mantle layer;PLR,pallidal
(D) Enlarged portion of (C) showing the overgrown choroid plexus.
ridge; sopt, optic sulcus; ven, ventricular layer.
mtl, mantle layer; ven, ventricular layer.
(L)Transversesection throughthe fourthventricle ofan e14.5homo-
(E) Nissl-stainedsection throughthebrainstem of ane16.5 homozy- zygous embryo reacted for lacZ activity detection. The choroid
gous fetus. 1, pituitary gland; 2, reticular nucleus; 3, facial nucleus; plexus shows overgrowth and intense Apaf1 expression. 4V, fourth
4, principal nucleus of the trigeminal; 5, eighth cranial nerve; 6, ventricle; chp, choroid plexus.
cochlear nuclei; 7, cerebellum. (M) Transverse section through the hindbrain of an e10.5 heterozy-
(F and G) Hematoxylin-eosin-stained sections through the cortex of gous mouse embryo reacted for lacZ activity detection. Apaf1 is
e14.5 embryos, homozygous (F) and wild-type (G) for the Apaf1 expressed in the marginal layer. flp, floor plate; hrp, hindbrain roof
mutation. The thickness and organization of the ventricular layer
plate; mar, marginal layer; ven, ventricular layer.

Apaf1 Regulates Apoptosis in Mouse Development
733
Figure 6. Susceptibility to Apoptosis of Apaf1
2
/
2
EFs
(A) Histogram showing the cell death percentage after 8 hr of
treatment.
(B) Histogram showing the cell death percentage after 20 hr of
treatment. The red line on top of the staurosporin/wild-type bar
emphasizes the fact that the full effect of the inducer (100% cell
death) was reached shortly after the first 8 hr (see [A]).
White, Apaf1 mutant cells; gray, wild-type cells. a-Fas, anti-Fas
antibody; C6-cer, C6-ceramide; STS, staurosporin.
al., 1996, 1998; this work). These in vivo results confirm
the invitro reconstitutionstudiesontheApaf1apoptotic
cascade and further emphasize the parallel apoptotic
Figure 5. The Apaf1 Mutant Tissues Contain No Activated Casp3
pathways existing in C. elegans and mammals (Li et al.,
Immunodetection of activated Casp3 by the CM1 antibody in the
1997; Zou et al., 1997).
telencephalon (A–D) and brain stem (E–H) of wild-type (A, B, E, and
F) and Apaf1 mutant (C, D, G, and H) e12.5 embryos. Activated
Interestingly, although Bax deficiency dramatically de-
Casp3–containing cells can be easily identified in the wild-type tis-
creases neuronal programmed cell death, unlike Apaf1,
sue, but not in the mutant. Four pairs of images are shown corre-
Casp9, and Casp3-deficient embryos, brainovergrowth
sponding to four sections illuminated with differentially filtered UV
is not a feature of the Bax
2
/
2
nervous system (Knudson
light in order to demonstrate the specific fluorescent signal due to
et al., 1995). Thus, Bax and BclX
L
may predominantly
the antibody detection (A, C,E, andG) and thesignal in the context
play a role in postmitotic neurons while the other mole-
of the tissue nonspecificallystained with bisbenzimide (B, D, F, and
H). Arrowheads in (A), (B), (E), and (F) show examples of labeled
cules in this apoptotic pathway act on both neuronal
cells in the wild-type tissue. Arrows in (E) and (F) show a labeled
progenitors and postmitotic cells.
neuritic process. The brightness of (C) and (G) has been increased
25% with respect to the rest of the photographs in the panel.
Apaf1 Mutant Embryonic Fibroblasts Are Less
Susceptible to Apoptotic Stimuli
To demonstrate the defect of apoptosis as the basis ofactivation in neurons undergoing programmed cell death;
CM1 immunoreactivity is markedly increased in BclX
L
- this extensive phenotype, we examinedthe susceptibil-
ity of Apaf1 homozygous embryonic fibroblasts (EFs),deficient embryos and decreased in Bax-deficient em-
bryonic brain (Srinivasan et al., 1998). Recently, it has dissected from e13.5 embryos, to three different apo-
ptotic stimuli in a time course experiment. Apaf1 homo-been reported that, as happens in Apaf1 homozygous
mutants and in Bax-deficient embryos, Casp9-deficient zygous and wild-type EFs showed the expected diver-
gent response to induction of apoptosis by anti-Fasmice had little CM1 immunoreactivity in the developing
nervous system and failed toactivate Casp3 in a variety antibody, C6-ceramide, and staurosporin, as revealed
by morphological analysis (Figure 6).ofexperimentalparadigms(Kuidaetal.,1998).Together,
these results indicate an in vivo apoptotic pathway The results can be summarized as follows: (1) After 8
hr of treatment with RMF2 anti-Fas antibody, no differ-whereby apoptotic stimulitrigger apoptosis,depending
on the ratio of Bax:BclX
L
in neurons,through amultimo- encewas observedbetweenwild-typeand homozygous
EFscelldeathrate(Figure6A).Nevertheless,aftera 20hrlecularcomplexcriticallyinvolving Apaf1andCasp9and
ultimately leading to Casp3 activation and apoptosis. long treatment, virtually all thewild-type EFsunderwent
apoptosis, while almost half of the Apaf1 mutant EFsDisruption of any of the proapoptotic molecules in this
pathway (Bax, Apaf1, Casp9, or Casp3) blocks the apo- were still alive (Figure 6B). (2) After an 8 hr long cell
treatment with C6-ceramide, the second messenger inptoticcascadeand leadstodramaticallydecreasedpro-
grammed cell death (Deckwerth et al., 1996; Kuida et the sphingomyelin pathway, cell death was absent in

Cell
734
the Apaf1 mutant EFs, while apoptosis induction was
effective in about 50% of the wild-typecells (Figure 6A).
(3) Likewise, after 8 hr of treatment, staurosporin (STS),
a broad-spectrum inhibitor of protein kinases that pre-
sumably acts downstream of receptors in signal trans-
duction processes, induced cell death in about 95% of
wild-type EFs but in only 30% of Apaf1
2
/
2
EFs (Figure
6A). Following prolonged treatment with both these re-
agents (C6-ceramideand STS),the differentialresponse
was proportionally confirmed (Figure 6B).
Ithasbeenshownthatapoptoticdefectsdue toCasp3
deficiency are remarkably stimulus-specific even within
the same cell type, including EFs (Woo et al., 1998).
Our results indicate that upstream pathways leading to
Casp3activation canbedistinctand/or cross-communi-
cating. For instance, the partial protective effect of
Apaf1 deficiency toFas-mediated apoptosis can be ac-
counted for by the hypothesized cross-communication
amongtwopathwaysofCasp3activation,mitochondrial
and Fas-mediated (Cryns and Yuan, 1998). Differential
response can be caused as well by other Casp3-like
proteases(Kuida etal.,1996;Wooet al.,1998).Similarly,
the existence of other Apaf1-like genes cannot be ex-
cluded. These conclusions havebeen confirmed by Yo-
shida et al. (1998 [this issue of Cell]).
APAF1 Is a Candidate Gene Involved
Figure 7. Chromosomal Localization ofApaf1 inMouse and Human
in Noonan Syndrome
(A and B) Example of FISH mapping results for probe pφ13.1 on
To test the possibility that the Apaf1 gene was located
mouse (A) and human (B) chromosomes. In both, the left panel
at sitesof mouse spontaneous mutationsand/or atsites
shows the FISH signals on the chromosome, and the right panel
associated with human syndromes, we mapped the
shows the same mitotic figure stained with DAPI to identify mouse
Apaf1 geneusing fluorescentin situhybridization (FISH)
chromosome 10 (A) and human chromosome 12 (B). Apaf1 probe
mapson mousechromosome 10,region C3-D1and onhuman chro-
on normal mouse and human chromosome spreads. An
mosome 12, q22-q23.
Apaf1 cDNA probe mapped on mouse chromosome 10,
region C3-D1 and on human chromosome 12, q22-q23
(Figure 7). These resultsare in accordance with the pre-
brain), morphogenetic cell death (in the neural tube,
dicted syntenicrelationship between humanand mouse
lens,skull, face, and limbs), and phylogeneticcell death
chromosomes that share a region of homology in the
(elimination of the hyaloid artery system in the devel-
distal part of their long arm (Copeland et al., 1990). This
oping eye). Furthermore, Apaf1 is required for Casp3
region isassociated in mouse withforebrainovergrowth
activation in embryonic brain in vivo.
(fog),a spontaneous autosomal recessive mutationpro-
It has been described that APAF1, BclX
L
, and CASP9
ducing excessive growthor cellularproliferation in fore-
formin vitroa ternarycomplex,as wellas theirhomologs
brain and midline cleft (Harris et al., 1997).
CED-4, CED-9, and CED-3 in C. elegans (Pan et al.,
The human autosomal dominant Noonan syndrome
1998), and that APAF1 is upstream of CASP3 (Zou et
(NS) has been associated with the region q22-qter of
al.,1997).Targeted mutationsin BclX,Casp3,and Casp9
the long arm of human chromosome 12 (Jamieson et
genes and the Apaf1 mutation we generated induce
al., 1994). So far no obvious candidate gene for NS
major abnormalities in brain development, respectively
has been identified. This syndrome has a complicated
due to excess and lack of cell death (Motoyama et al.,
clinical synopsis, mainly characterized by typical facial
1995; Kuida et al., 1996, 1998; this work; Yoshida et al.,
abnormalities, congenital cardiacdefects,and limb mal-
1998); these similarities together with the finding that
formations. In some casesthepatients also show retinal
Apaf1 is upstream to Casp3 in the apoptotic cascade
degenerations as retinitis pigmentosa, which has been
imply thepossibility of a functional connectionbetween
shown to bea degenerative diseaserelatedtoapoptosis
Apaf1, BclX
L
, Casp9, and Casp3 during mouse devel-
defect (Wong, 1994; Lorenzetti and Fryns, 1996). There-
opment.
fore, APAF1 can be considered as a candidate to be
Nevertheless, in this study we haveshown that Apaf1
involved in NS.
is required in several other apoptotic processes in mu-
rine development and in multiple cell types. Our results
imply that Apaf1 can be a key regulator of other apo-Concluding Remarks
Apaf1, a key regulator of the apoptotic pathway, has a ptotic pathways, involving other caspases and/or Bcl2-
like proteins, and raise the possibility that deficienciesmajor role in developmental programmed cell death.
Specifically, Apaf1 is involved in histogenetic cell death in distinct developmental programmed cell death path-
ways can account for distinct phenotypes.(control of cell number in the developing retina and

Apaf1 Regulates Apoptosis in Mouse Development
735
Eagle’s medium (DMEM) supplemented with 5% fetal calf serum
Finally, the phenotype of the Apaf1 mutant mice to-
and gentamycin, and plated.Twenty-four hours after the initial plat-
getherwiththe chromosomallocalizationof APAF1gene
ing, the cells were subjected to the following treatments at 378C for
in humans renders thisgene a candidate to be mutated
20 hr:anti-Fas antibody(RMF2 1 mg/ml, Immunotech),C6-ceramide
in Noonan syndrome.
(30 mM, BIOMOL), and Staurosporin(2 mM, BIOMOL). Pretreatment
of the plated cells with 10 mg/ml cycloheximide was carried out in
Experimental Procedures order to inhibit protein synthesis (Weil et al., 1996).
The cells were then analyzed by morphological criteria, and apo-
Generation of Apaf1-Deficient Mice and Cloning ptotic events were counted after 8 hr and after 20 hr of treatment
of Murine Apaf1 cDNA
over ten photographic fields. The percentage of dying cells was
ES celllineGTXIX-18, containinganinsertion ofthe genetrap vector
calculated in comparison with a negative control plate.
IRESbgeo within the Apaf1 gene, was generated as described
(Chowdhury et al., 1997). The 59RACE on ES cells RNA yielded 505
Chromosomal Localization by FISH
bp, upstream of the insertional splice acceptor site, that matched
The pφ13.1 probe was labeled by nick translation using the BRL
human APAF1 sequence with 78% of nucleotide identities (Zou et
BioNick labeling kit(158C,1hr),andtheprocedure forFISH detection
al., 1997). This 505bp long fragment was used asa probe toscreen
was performed according to Heng et al. (1992) and Heng and Tsui
an e15.5 mouse cDNA library (Clontech), yielding several overlap-
(1993).
ping clones that were sequenced. A final consensus 5227 bp long,
FISH signals and the DAPI banding pattern were recorded sepa-
containing the entire 59UTR and coding regions and partial 39UTR
rately by taking photographs, and theassignment of the FISHmap-
regions of murine Apaf1 cDNA, was obtained.
ping datawith chromosomalbands was achievedby superimposing
Founder chimeric males, generated as described from 129/Sv-
FISHsignals withDAPIbandedchromosomes(Heng andTsui, 1993).
derived ES cells (Chowdhury et al., 1997), were mated with outbred
Under the conditions used, the hybridization efficiency was ap-
NMRIfemales, and heterozygousprogenies weremated to maintain
proximately 67% for this probe on the mouse chromosomes and
the allele.
77% on the human chromosomes. After chromosome assignment,
the detailed position was furtherdetermined based on the summary
Southern, Northern, and Western Analyses
from ten photos.
A probe for lacZ sequences (LZ) was synthesized from an AvaI
fragment of pCH110 (Pharmacia). A 213 bp long probe specific for
Acknowledgments
the region immediately upstream to the insertional splice acceptor
site (p13.02) spreading between position 2387 and 2600 of the
We thank Dr. M. Kessel, Dr. A. Mansouri, Dr. A. Stoykova, Dr. G.
mouse Apaf1cDNA was synthesized digesting plasmid pφ13.1(3.2
Bernier, Dr. M. Salminen, Dr. M. Schwarz, J. Berger, A. De Antoni,
kb) with PstI. Probes LZ and p13.02 were used for genotyping by
and A. Pires for comments and suggestions. We are indebted to
Southern blot analysis.
Dr. K.Chowdhury fortheIRESbgeo vector. WethankDrM.Piacentini
RNA blots containing 2 mg RNA per lane from different mouse
(Rome) forhelpfuldiscussion.We acknowledgethe excellenttechni-
adult tissues and embryonic stages were purchased from Clontech
cal assistanceofR.Altscha
¨
ffel,A.Ficner,S.Hille, R.Libal,S.Mahsur,
and probed with pφ13.1. RNA poly(A)1 from Apaf1
2
/
2
, Apaf
1
/
2
,
S. Schlott, T. Schulz, and A. Voigt. We also thank Dr. A. Srinivasan
and Apaf
1
/
1
embryos was hybridized with pφ13.1. Southern and
(IDUN Pharmaceuticals) for the generous gift of CM1 antiserum.
Northern blotswere hybridized witha 1.4kb Fkh-5 cDNAprobe and
Thanks are due to SeqLab (Go
¨
ttingen) and seeDNA (Toronto) for
a 2.0 kb b-actin cDNA probe, respectively, as internal controls.
DNA sequencing and chromosomal localization, respectively. This
Protein extracts were prepared from Apaf1
2
/
2
, Apaf1
1
/
2
, and
project was supported by Amgen, Inc. (ThousandOaks, CA), by the
Apaf
1
/
1
e10.5 brains, and Western blot analysis was performed
Deutsche Forschungsgemeinschaft Leibniz-Program, and by the
using anti-APAF1 antibody (Santa Cruz Biotechnology) according
Max PlanckSociety.F. C.is supported bya HumanFrontier Science
to manufacturer’s instructions.
Project Fellowship, and G. A.-B. is supported by a European Union
Fellowship. K. A. R.’s lab is supported by National Institutes of
Histological and Immunocytochemical Analyses
Health grants NS35107 and NS35484.
Whole-mountlacZstainingofembryos atdifferentembryonicstages
was performed as described (Gossler et al., 1989). For albumin-
gelatinsectioning, thetreated embryos wereembedded inalbumin-
Received May 21, 1998; revised August 3, 1998.
gelatinand vibratomesectioned into slices 40mm thick.For paraffin
sectioning specimens were impregnated with Paraffin wax (Para-
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GenBank Accession Number
The accessionnumber forthe nucleotide sequenceofmurine Apaf1
is AF064071.