Apoptosis (2010) 15:331349
DOI 10.1007/s10495-009-0432-9
UNUSUAL MODEL SYSTEMS FOR CELL DEATH RESEARCH
The zebrafish as a model organism for the study of apoptosis
Peter M. Eimon Avi Ashkenazi
Published online: 24 December 2009
Springer Science+Business Media, LLC 2009
Abstract Apoptosis plays important roles in embryo-
genesis, tissue homeostasis, and immune system regula-
tion. The zebrafish (Danio rerio) is a powerful vertebrate
model organism that has been extensively used to study
apoptotic cell death during normal development and under
conditions of cellular stress. In the past 5 years, a detailed
picture has begun to emerge of the molecular underpin-
nings of the cell-intrinsic and the cell-extrinsic apoptosis
signaling pathways in zebrafish. We begin this review with
an introduction to the techniques and experimental
approaches that are used to study apoptosis in zebrafish.
We follow with a general overview of developmental
apoptosis during zebrafish embryogenesis. Finally, we
present a comprehensive review of the intrinsic and
extrinsic apoptosis pathways in zebrafish, focusing on the
high degree of conservation with humans and other mam-
mals. Recent publications that draw upon the unique
advantages of the zebrafish system to study novel aspects
of apoptosis regulation and function are highlighted
throughout.
Keywords Apoptosis
Zebrafish
Intrinsic pathway

Extrinsic pathway
Bcl-2
IAP
Death receptor

Apo2L/TRAIL
Caspase
P. M. Eimon
Zygogen, LLC, Atlanta, GA, USA
A. Ashkenazi (&)
Department of Molecular Oncology, Genentech Inc.,
1 DNA Way, MS 42, South San Francisco, CA 94080, USA
e-mail: aa@gene.com
Introduction
There are two key apoptotic signaling mechanisms in
mammals and other vertebrates: the cell-intrinsic (or
mitochondrial) pathway and the cell-extrinsic (or death
receptor) pathway. The intrinsic pathway can be triggered
by a variety of cues including cellular damage, growth
factor withdrawal, and chemotherapeutic drugs. It is reg-
ulated by the Bcl-2 gene family and functions through the
initiator protease caspase-9 [1]. The extrinsic pathway
plays important roles in immune system operation and
homeostasis. It is triggered by death receptor ligation and
functions through a death-inducing signaling complex
(DISC) that includes the initiator proteases caspase-8 and
-10 [2]. The extrinsic pathway is linked to the intrinsic
pathway through Bid, a Bcl-2 family protein that can be
cleaved and activated by caspase-8. Additional pathways,
such as the granzyme B and endoplasmic reticulum path-
ways, may also mediate aspects of apoptosis under certain
conditions. All pathways converge at the level of the
effector caspases (caspase-3, -6 and -7), which carry out
the molecular mechanics of programmed cell death [3].
The intrinsic apoptosis pathway appears to have evolved
around the same time as multicellular organisms, and Bcl-2
family orthologs have been identified in all metazoans
analyzed to date [1]. In contrast, the extrinsic pathway is a
more recent evolutionary development. No tumor necrosis
factor (TNF) or TNF receptor (TNFR) superfamily mem-
bers have been found in Caenorhabditis elegans (C. ele-
gans). Death receptors (TNFRs possessing a cytoplasmic
death domain motif) have been reported almost exclusively
in vertebrates, although recently a death domain-containing
TNFR has been identified in the Zhikong scallop (Chlamys
farreri) [4]. In the following review we will summarize the
current understanding of the intrinsic and extrinsic
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apoptotic pathways in zebrafish. We will highlight simi-
larities and differences between these pathways in zebra-
fish, mammals, and more divergent model organisms such
as fruit flies and nematodes. In order to promote consis-
tency in zebrafish nomenclature, we have used the official
gene names and symbols endorsed by Entrez and the
Zebrafish Information Network (ZFIN) throughout this
review. In some cases, official zebrafish gene names are
paired with their more familiar human designation (e.g.
tnfrsf21/dr6). LocusLink (LOC) designations are used for
zebrafish genes without an official full name.
Approaches to studying apoptosis in zebrafish
The zebrafish is a small freshwater teleost of the cyprinid
family (carps and minnows) in the class of ray-finned fishes
(Actinopterygii). Zebrafish have served as an important
research organism for decades, particularly in the fields of
genetics and developmental biology [5]. A unique combi-
nation of features makes them particularly attractive as a
vertebrate model. These include their small size (embryos
are *0.7 mm in diameter at fertilization and grow to
3.5 mm within 3 days), high fecundity (a single zebrafish
pair can produce hundreds of embryos per week), short
generation time (adults reach sexual maturity
in *3 months), and genetic tractability. Embryos are fer-
tilized externally, undergo rapid and synchronous devel-
opment, and are optically transparent. Most organs become
functional between 3 and 5 days post fertilization (dpf) [6].
The popularity of zebrafish increased substantially fol-
lowing several publications in 1996 demonstrating that
they are amenable to large-scale forward genetic screens
and describing over 1000 mutants with visible phenotypes
during early development [7, 8]. Previously, large-scale
screening approaches had been limited to invertebrates
such as C. elegans and Drosophila melanogaster.
Many additional tools further enhance the utility of
zebrafish as a model organism. Reverse genetic approaches
include using antisense morpholino oligonucleotides to
knock down gene function transiently in embryos [9], tar-
geting induced local lesions in genomes (TILLING) to
identify stable mutations in genes of interest [10], and zinc-
finger nucleases to generate locus-specific mutations for
targeted gene inactivation [11]. Techniques have been
devised for generating stable transgenic lines that express
fluorescent reporter proteins or other genes of interest
under the control of tissue-specific promoters [1214].
Commercial zebrafish DNA microarrays are available for
expression profiling, and protocols have been developed
for proteomic and metabolomic analysis [15, 16]. The
Sanger Institute has sequenced the zebrafish genome
and an annotated assembly is available in Ensembl
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Apoptosis (2010) 15:331349
(http://www.ensembl.org/Danio_rerio/Info/Index). More com-
plete manual annotation is being carried out in collabora-
tion with ZFIN and can be accessed through the Vega
database (http://vega.sanger.ac.uk/Danio_rerio/index.html).
As a result of these and other approaches, an exceptionally
large number of genes and molecular pathways have been
well characterized in zebrafish and help to validate this
model organism as highly relevant for the study of verte-
brate biology.
Several experimental protocols have been developed
that allow apoptotic cells to be visualized in vivo in zeb-
rafish embryos. One of the most commonly used tech-
niques utilizes the vital dye acridine orange (AO), a nucleic
acid intercalating dye that emits green fluorescence when
bound to dsDNA and selectively stains apoptotic rather
than necrotic cells [17, 18]. Analysis of AO staining in
zebrafish embryos is complicated by autofluorescence or
non-specific uptake in the yolk, iridophores, hatching
gland, and neuromasts [19]. A recent publication describes
an imaging protocol that enables rapid quantification of AO
staining in large groups of embryos [20]. Approaches such
as this are crucial, since AO staining exhibits considerable
embryo-to-embryo variability and large numbers of
embryos must be analyzed to ensure statistical rigor.
Hoechst 33342, a DNA-binding vital dye, has also been
used in live zebrafish embryos to visualize the formation of
chromatin clumps characteristic of apoptotic nuclei [21].
Finally, a transgenic zebrafish line expressing mitochond-
rially-targeted green fluorescent protein (GFP) has been
developed to monitor mitochondrial fragmentation in
apoptotic cells in vivo [22].
Many assays used to detect apoptosis in mammalian
tissue samples and cell culture have been adapted to fixed
zebrafish embryos and embryonic lysates. Fixed embryos
can be stained by the terminal dUTP nick end-labeling
(TUNEL) method, which detects fragmented DNA char-
acteristic of apoptotic cells [18, 23]. According to some
reports, use of the TUNEL method on zebrafish embryos
older than 48 h post fertilization (hpf) is hampered by
unacceptable background staining and nonspecific labeling
of periderm cells [19]. Fixed embryos can also be immu-
stained using a monoclonal antibody that recognizes the
cleaved, active form of caspase-3 [24]. Fluorogenic and
bioluminescent substrates have been used to monitor the
activity of caspase-3, caspase-8, and caspase-9 in zebrafish
embryo lysates and in dissociated cells [21, 25, 26].
Negron and Lockshin [21] have used a number of
additional techniques to characterize the biochemical
modifications associated with apoptotic cell death in zeb-
rafish embryos and report broad conservation of apoptotic
hallmarks between fish and mammals. Extensive intranu-
cleosomal DNA fragmentation was confirmed by agarose
gel electrophoresis and poly(ADP-ribose) polymerase
Apoptosis (2010) 15:331349
(PARP) cleavage was detected by Western blot. Apoptotic
cells translocated phosphatidylserinea component of the
apoptotic cell-surface eat-me signalto the outer leaflet apoptotic cell death has been reported in mammalian
of the plasma membrane. Transmission electron micros-
copy was used to confirm that apoptotic cells in zebrafish
exhibit chromatin condensation and convolution of the
nuclear membrane. Engulfment of apoptotic cell corpses by
neighboring cells has also been observed in zebrafish
embryos using electron microscopy; engulfment can be
inhibited by knockdown of the phosphatidylserine receptor
jmjd6 [27]. In zebrafish, like mammals, apoptosis and
necrosis share a number of common features and it is
important to use two or more distinct assays to confirm that
cell death is truly apoptotic.
Many experimental methods for inducing apoptosis in
mammalian cells are also effective in zebrafish. Chemical
reagents that have been evaluated include aphidicolin [28],
camptothecin [28, 29], cycloheximide [21], hydroxyurea
[28], and staurosporine [22]. Apoptotic cell death has also
been demonstrated in response to heat shock [30], ultra-
violet irradiation [29], and c-irradiation [3032]. Similar to
mammals, stress-induced apoptosis in zebrafish can be
blocked by the pan-caspase inhibitor Z-VAD-FMK and by
the cytoprotective agent amifostine [26].
Developmental apoptosis in zebrafish
In multicellular organisms, normal development depends
upon a carefully orchestrated balance between cell prolif-
eration, differentiation, and death. Apoptosis is the most
common mechanism employed during development to
remove damaged, misplaced, or otherwise unwanted cells
[33, 34]. Additional forms of programmed cell death
particularly autophagy (or type II cell death)are utilized
in certain developmental contexts but fall outside the scope
of this review. Although over-production of cells that will
ultimately be eliminated by apoptosis may appear ener-
getically wasteful, it is a common feature of metazoan
development and occurs in species ranging from nema-
todes to humans [34, 35].
Zebrafish embryos are resistant to apoptosis before they
reach the maternal-to-zygotic transition (MZT, occurring
between 4 and 7 hpf), the stage during early development
when the zygotic genome becomes the primary source of
mRNA in the embryo [21, 28, 36]. Embryos treated with
camptothecin, cycloheximide, nocodazole, or staurosporine
prior to the end of MZT exhibit an arrest of cell prolifer-
ation but survive until the mid-gastrula stage (*7 hpf), at
which point they undergo rapid and synchronous cell death.
Acquisition of apoptotic competence appears to be under
post-translational control, as it is not impacted by contin-
uous treatment with cycloheximide. A similar inhibition of
333
the apoptotic machinery in pre-gastrula stage embryos has
been documented in Xenopus laevis [37]. In contrast,
embryos as early as the 16-cell stage [34, 38].
Following gastrulation, apoptotic cells are detected in
various organs and tissues of zebrafish embryos [19]. Cole
and Ross [39] have used the TUNEL method to map
developmental apoptosis in zebrafish between gastrulation
and 4 dpf. The authors describe transient high levels of
developmental apoptosis in a number of structures
including the olfactory placodes (2272 hpf), eyes (peak-
ing at 36 hpf in the retina and 2048 hpf in the lens), lat-
eral line neuromasts (beginning at 2430 hpf), Rohon-
Beard neurons (peaking between 3648 hpf), trigeminal
neurons (peaking between 3648 hpf), urogenital system
(peaking between 3036 hpf), and tail bud (peaking at
29 hpf). Similar patterns of developmental apoptosis have
been observed in other fish species such as medaka
(Oryzias latipes) [40].
Developmental apoptosis has been studied in detail in
several zebrafish neuronal populations. Cell death is
widespread in the central and peripheral nervous systems
of most vertebrates during development. In many regions,
more than 50% of newly formed neurons are eliminated
[41]. In motoneurons, one well-characterized period of
apoptosis occurs when axons contact their postsynaptic
targets. Competition between neurons for limiting amounts
of muscle-derived neurotrophic factors is thought to be a
major driver of apoptosis in this context [42]. Eisen and
Melancon [43] have used zebrafish embryos to study the
regulation of motoneuron apoptosis in greater detail. In
adult zebrafish, each ventral myotome is innervated by a
single motoneuron designated CaP. During early develop-
ment, however, a second motoneuron (designated VaP) is
often present alongside CaP in the spinal cord. Both CaP
and VaP are morphologically equivalent and send out
axons that pause briefly at an intermediate target known as
the horizontal myoseptum (HM). The cell whose axon first
reaches the HM adopts the CaP fate and goes on to
innervate the ventral myotome. The remaining cell adopts
the VaP fate, its axon remains paused at the HM, and it
undergoes apoptotic death by 36 hpf. Through a series of
cell transplant and cell ablation studies, the authors show
that VaP motoneuron death is governed by a death-pro-
moting signal originating from HM cells and triggered by
the CaP axon [43, 44]. Developmental apoptosis is also
widespread among neurons of the mammalian retina,
where two consecutive phases of apoptosis have been
described. The earlier phase occurs during neurogenesis
and cell migration, while the later phase occurs during
target innervation [45, 46]. A similar pattern of apoptosis is
seen in the developing zebrafish retina, with an initial
phase peaking at 34 dpf and a second phase at 67 dpf
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[47]. The first phase is restricted to the ganglion cell layer
(GCL) and the inner nuclear layer (INL), while the second
phase encompasses cells of outer nuclear layer (ONL) in
addition to the GCL and INL. Although the onset and
progression of retinal apoptosis in zebrafish is reminiscent
of mammals, the total number of apoptotic cells detected in
zebrafish is significantly less.
Developmental apoptosis in non-neuronal cell types has
also been characterized in zebrafish. Elevated levels of
apoptotic cells are detected in the zebrafish lens around
24 hpf as the developing lens is detaching from the surface
ectoderm. It has been suggested that apoptosis aids in
separation of the lens from the ectoderm [48], similar to a
mechanism reported during development of the rat lens
vesicle [49]. Two more recent studies have failed to con-
firm that apoptosis is a significant factor in separation of
the zebrafish lens from the surface ectoderm [50, 51].
Apoptosis may instead be used to clear undifferentiated or
lens-foreign cells that could interfere with subsequent
development [48, 51]. TUNEL-positive cells in the lens
may also arise from programmed removal of nuclei and
other organelles from lens fiber cells, a process that shares
many hallmarks of apoptosis including chromatin frag-
mentation [52].
In some cases, developmental apoptosis underlies pro-
cesses that have not been evolutionarily conserved between
zebrafish and other vertebrates. Rohon-Beard (RB) cells
are an interesting example of this phenomenon. These
primary sensory neurons are found in anamniote verte-
brates such as teleosts and amphibians but are not present
in birds or mammals. RB cells exist only transiently during
early development and their sensory functions are soon
replaced by neurons of the dorsal root ganglia. In zebrafish,
RB cells are eliminated during the first 4 days of devel-
opment in a caspase-dependent manner. Apoptosis appears
to be driven in part by loss of the neurotrophic factor ntf3
and may also be regulated by voltage-gated Na? current
[5355]. The inner ear is another region where the role of
developmental apoptosis has not been conserved through-
out vertebrate evolution. In both birds and mammals an
apoptotic hot spot has been characterized in the ven- bers are categorized as multidomain proteins (containing
tromedial wall of the developing inner ear adjacent to the
auditory ganglion [56]. A thorough examination of apop-
totic cells in the developing ears of both zebrafish and
Xenopus laevis failed to detect a similar hot spot, although
apoptosis was observed in the statoacoustic ganglion in
agreement with previous reports in birds and mammals
[57].
Large-scale mutagenesis screens in zebrafish have
identified numerous genes with loss-of-function pheno-
types that affect developmental apoptosis. By far the
largest class of these mutants is associated with elevated
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Apoptosis (2010) 15:331349
cell death in the central nervous system (CNS; for exam-
ples, see Abdelilah et al. [58], Furutani-Seiki et al. [18],
Fadool et al. [59], and Rodriguez and Driever [19]).
Mutations that affect cell death in many other organs and
tissues have also been described (reviewed in Pyati et al.
[60]). It is probable that aberrant cell death in most of these
mutants does not stem from defects in the core apoptotic
machinery, but rather serves to eliminate cells that are
unhealthy, misplaced, or otherwise deficient due to dis-
ruptions in the tightly regulated process of embryogenesis.
In addition to forward genetic screens, knockdown exper-
iments have suggested that many zebrafish genes have
loss-of-function phenotypes characterized by excessive
apoptotic cell death (reviewed in Pyati et al. [60]).
Unfortunately, interpretation of these results is greatly
complicated by recent findings that morpholino antisense
oligonucleotides and other knockdown technologies often
induce nonspecific off-target apoptosis in zebrafish [61].
Off-target apoptosis is most pronounced in the CNS and
appears to be mediated through tp53 activation. For a more
detailed discussion of mutants and morpholino knockdown
embryos in which developmental cell death is perturbed,
readers are encouraged to consult Pyati et al. [60]. The
remainder of this review will focus specifically on the
molecular mechanism governing the intrinsic and extrinsic
apoptosis pathways in zebrafish.
The cell-intrinsic apoptosis pathway
The zebrafish Bcl-2 family
The Bcl-2 gene family serves as the chief regulator of
the intrinsic apoptosis pathway in mammals [6264]. The
Bcl-2 family can be divided into three subfamilies based
on apoptotic function and the presence of up to four distinct
a-helical motifs known as Bcl-2 homology (BH) domains
(designated BH1, BH2, BH3, and BH4). Anti-apoptotic
family members (e.g. Bcl-2, Bcl-xL, Bcl-w, and Mcl-1)
possess all four BH domains. Pro-apoptotic family mem-
BH1, BH2, and BH3 domains; e.g. Bax, Bak, and Bok) and
BH3-only proteins (e.g. Bid, Bad, Bik, Bim, Bmf, Noxa,
Puma, Hrk, and the Bnip proteins). Members of the BH3-
only subfamily serve as apical sensors for various apoptotic
stimuli and are capable of promoting apoptosis through
activation of the multidomain pro-apoptotic subfamily and/
or suppression of the anti-apoptotic subfamily. Interactions
between subfamily members occur when the BH3 domains
on pro-apoptotic proteins bind to a hydrophobic groove
formed by the BH1, BH2, and BH3 domains on anti-
apoptotic proteins [65].
Apoptosis (2010) 15:331349 335

Table 1 Apoptosis associated genes in zebrafish

Gene family Mammalian gene Zebrafish Other names Reference Zebrafish Synteny
homologa sequence chromosome w/human

TNF LTa (TNFSF1)

TNF (TNFSF2) tnfa TNF1, tnf-alpha, NM_212859 19 Yes [172]
TNF-a_v1, TNF-a_v3
TNF (TNFSF2) tnfb TNF2, tnf, Tnf-alpha, NM_001024447 15 Yes [24]
TNF-a_v2
CD95L/FasL (TNFSF6) faslg FasL NM_001042701 16
Apo2L/TRAIL (TNFSF10) tnfsf10l DL1a, TRAIL-like NM_131843 7
Apo2L/TRAIL (TNFSF10) tnfsf10l2 DL2 NM_001002593 24 Yes [24]
Apo2L/TRAIL (TNFSF10) tnfsf10l3 DL1b NM_001042713 21
Apo2L/TRAIL (TNFSF10) tnfsf10l4 DL3 NM_001013283 5
TL1A (TNFSF15) LOC560966 similar to TL1A NM_001123259 8
Unknown lta TNF-N NM_001024821 15
TNFR TNFR1 (TNFRSF1A) tnfrsf1a TNFR1 NM_213190 16 Yes [24]
CD95/Fas (TNFRSF6) fas XM_685355 17 Yes [24]
DR4 & DR5 hdr ZH-DR NM_194391 5
(TNFRSF10A & B)
DR4 & DR5 tnfrsfa OTR, tnf NM_131840 21
(TNFRSF10A & B)
DR6 (TNFRSF21) tnfrsf21 DR6 NM_001042688 20 Yes [24]
DR3 (TNFRSF25)
Adaptors CRADD/RAIDD cradd NM_001006066 4 Yes (ZFIN)
DEDD zgc:92202 NM_001002639 8
FADD fadd XM_001923858 7 Yes [24]
EDARADD LOC566761 similar to EDRADD XM_690046 17
MADD LOC100150917 similar to MADD XM_001923955 7
PIDD/LRDD LOC571033 similar to LRDD XM_694585 25
RIP1/RIPK1 ripk1l rip1 NM_001043350 2
TRADD tradd NM_131607 7 Yes (ZFIN)
Caspase Unknown caspase-a caspy NM_131505 16
Unknown caspase-b caspy2 NM_152884 1
Caspase-2 caspase-2 NM_001042695 16 Yes [24]
Caspase-3 caspase-3a casp3 NM_131877 1 Yes [24]
Caspase-3 caspase-3b casp3l NM_001048066 7 Yes [24]
Caspase-6 caspase-6 casp6a NM_001020497 3
Caspase-6 caspase-6l1 casp6b NM_001005973 3
Caspase-6 caspase-6l2 casp6c NM_001039980 3
Caspase-7 caspase-7 casp7a NM_001020607 23 Yes [24]
Caspase-7 LOC100000522 casp7b, similar to Casp7 XM_001340668 20
Caspase-7 LOC798445 similar to Casp7 XM_001338854 10
Caspase-8 caspase-8 casp8a NM_131510 6 Yes [146]
Caspase-8 caspase-8l1 casp8a-like NM_001098619 16
Caspase-8 caspase-8l2 casp8b, casp10-like XM_680338 9
Caspase-9 caspase-9 NM_001007404 23 Yes [24]
Caspase-10 LOC795066 caspxb, similar to Casp10 XM_001335127 16
Caspase-10 caspase-xa NM_001083862 6
zgc:194469 card-casp8 NM_001136252 6
c-FLIP/CFLAR cflar FLIP, Clarp1 NM_194399 11

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Table 1 continued
Gene family Mammalian gene Zebrafish Other names Reference Zebrafish Synteny
homologa sequence chromosome w/human

Bcl2 Bcl-2 bcl2 zBlp2 NM_001030253 24

(Pro-Survival) Bcl-xL (BCL2L1) bcl2l zBlp1, zfBLP1, zfBcl-xL NM_131807 23 Yes [31]
Bcl-w (BCL2L2)
Mcl-1 mcl1a zfMLP1, zfMcl-1a NM_131599 19 Yes [31]
Mcl-1 mcl1b NM_194394 16 Yes [31]
BCL2L10 bcl2l10 zNR13, Nrz, NR-13, mcl1l NM_194398 18 Yes [31]
Bfl-1 (BCL2A1)
BCL2L12
Bcl2 Bax baxa zBax1, bax NM_131562 3 Yes (ZFIN)
(Pro-Apoptotic) Bax baxb zBax2 NM_001013296 3 Yes [31]
Bak
Bok boka zBok1, bok NM_001003612 6 Yes [31]
Bok bokb zBok2 NM_201185 2 Yes [31]
Bcl-G (BCL2L14)
Bcl-rambo (BCL2L13) bcl2l13 NM_001044891 18 Yes (ZFIN)
Bfk (BCL2L15)
Bid bida zBid NM_001079826 18
Bad bad NM_131579 7 Yes [31]
Bik bik NM_001045038 4 Yes [31]
Bim (BCL2L11) bcl2l11 BIM NM_001135791 13
Bmf bmf1 NM_001045224 3
Bmf bmf2 NM_001045473 Yes [31]
Bnip1 LOC795732 similar to Bnip1 XM_001333689 21
Bnip1 LOC565822 similar to SEC20 XM_684156 14
Bnip2 bnip2 NM_201218 Unmapped Yes (ZFIN)
Bnip2 LOC100149386 similar to Bnip2 NM_001128394 16
Bnip3 zgc:123120 nip3b NM_212693 17
Bnip3 bnip3l nip3a NM_205571 10 Yes (ZFIN)
Noxa/PMAIP1 pmaip1 zNoxa NM_001045474 14
Puma/BBC3 bbc3 zPuma NM_001045472 15 Yes [31]
Hrk
IAP NAIP
cIAP1 (BIRC2) birc2 IAP1, birc3 NM_194395 21 Yes (ZFIN)
cIAP2 (BIRC3)
XIAP (BIRC4) xiap birc4 NM_194396 14
Survivin (BIRC5) birc5a bir5a, survivin, survivin 1 NM_194397 12 Yes [136]
Survivin (BIRC5) birc5b survivin 2 NM_145195 23
Apollon (BIRC6) LOC796547 similar to birc6 XM_001336866 17
MLIAP (BIRC7)
ILP2 (BIRC8)
Other A20/TNFAIP3 LOC564497 similar to TNFAIP3 XM_687830 13
Apaf-1 apaf1 NM_001045243 4 Yes (ZFIN)
APP appa app NM_131564 1 Yes [136]
ATM atm XM_001921710 12
ATR DKEY- atr XM_691071 2
231J24.1

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Table 1 continued
Gene family Mammalian gene Zebrafish Other names Reference Zebrafish Synteny
homologa sequence chromosome w/human

CHK1/CHEK1 chek1 chk1 NM_200193 21

CHK2/CHEK2 chek2 chk2 NM_200045 5
COP1/RFWD2 zgc:163067 NM_001089542 2
Cullin 3 cullin 3a cullin 3 NM_199691 2
Cytochrome c zgc:86706 NM_001002068 6
Mdm2 mdm2 NM_131364 4 Yes [136]
Omi/HtrA2 LOC799006 XM_001339375 13
Omi/HtrA2 LOC100003308 NM_001109735 13
Omi/HtrA2 LOC560031 XM_702479 10
Omi/HtrA2 LOC797876 XM_001338303 13
Omi/HtrA2 LOC560704 XM_001921275 13
Omi/HtrA2 LOC560164 XM_001921286 13
Omi/HtrA2 LOC562245 XM_001921346 13
Omi/HtrA2 LOC797799 NM_001110528 13
Omi/HtrA2 LOC100148101 XM_001921090 21
Omi/HtrA2 LOC793110 XM_001332768 10
Omi/HtrA2 LOC793521 XM_001333269 10
Omi/HtrA2 zgc:174193 NM_001114706 13
Omi/HtrA2 LOC799537 NM_001110170 13
Omi/HtrA2 LOC799634 XM_001335165 21
Omi/HtrA2 LOC100150281 XM_001923368 21
Omi/HtrA2 LOC799293 XM_001922056 21
Omi/HtrA2 LOC100149563 NM_001128820 13
p21/CDKN1A cdkn1a p21, cip1, waf1 XM_001923789 22 Yes (ZFIN)
p53 tp53 p53, brp53, drp53 NM_131327 5 Yes [136]
p62/SQSTM1 sequestosome 1 NM_213173 14 Yes (ZFIN)b
p62/SQSTM1 LOC100148880 similar to Sequestosome 1 XM_001922985 14
Phosphatidylserine receptor jmjd6 psr, ptdsr, zfpsr NM_170761 3
Pirh2/RCHY1 rchy1 NM_212600 5
SMAC/DIABLO zgc:63938 NM_200346 15
SMAC/DIABLO zgc:158776 XM_693904 21
a
LocusLink (LOC) designations are used for genes without an official full name
b
Conserved synteny reported with non-human mammalian species

Orthologs of nearly every member of the Bcl-2 family
have been described in zebrafish (Table 1) [31, 62, 6671].
The only exceptions are three members of the anti-apop-
totic subfamily (Bcl-w, Bfl-1, and BCL2L12), one member
of the multidomain pro-apoptotic subfamily (Bak), and
three members of the BH3-only subfamily (Bcl-G, Bfk, and
Hrk). Zebrafish multidomain Bcl-2 family members show
the highest degree of conservation with their mammalian
counterparts. Members of the BH3-only subfamily display
greater sequence divergence, making conserved synteny
within the genome an important criterion for establishing
orthology [31, 66, 67]. It is probable that additional
zebrafish Bcl-2 proteins remain to be described, particu-
larly among members of the divergent BH3-only subfam-
ily. The wide range of Bcl-2 family members identified to
date suggests that zebrafish may be a better system for
modeling the mammalian intrinsic apoptosis pathway than
simple metazoans such as Drosophila, where only two
multidomain Bcl-2 proteins have been described (buffy and
debcl) and C. elegans, where one multidomain Bcl-2 pro-
tein (ced-9) and two BH3-only protein (ced-13 and egl-1)
have been described (Fig. 1) [62, 72, 73].
There is a great deal of functional conservation between
pro-apoptotic Bcl-2 proteins in zebrafish and mammals.

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Fig. 1 Comparison of Apoptotic Pathways in C. elegans, Drosophila,
Fish, and Mammals. The diagram illustrates core components of the
intrinsic and extrinsic apoptosis pathways in evolutionarily diverse
species. In zebrafish, red arrows indicate interactions with strong
supporting evidence based on co-immunoprecipitation studies and/or
antisense morpholino oligonucleotide knockdown experiments. In
some instances, interactions may not be established for all members
of a given family. For example, tnfsf10l, tnfsf10l2, and tnfsf10l3 have
Overexpression of zebrafish BH3-only subfamily members
bida, bik, bcl2l11/bim, bmf1, bmf2, pmaip1/noxa, bbc3/
puma, and bnip3l and the multidomain pro-apoptotic sub-
family members baxa and baxb in early stage embryos
induces caspase-3 activation and apoptosis [31, 66, 74].
Conversely, inhibition of bbc3/puma, baxa, or baxb using
antisense morpholino oligonucleotides protects embryos
from apoptosis in response to c-irradiation [31]. As in
mammals, the pro-apoptotic function of zebrafish BH3-only
proteins requires an intact BH3 domain. Mutation of this
domain blocks the ability of zebrafish bida, bcl2l11/bim,
pmaip1/noxa, and bbc3/puma to induce apoptosis in
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Apoptosis (2010) 15:331349
all been shown to trigger apoptosis through cell surface death
receptors but this activity has not yet been formally demonstrated for
faslg. Similarly, anti-apoptotic activity has been demonstrated for
birc2, birc5a, and birc5b but not the other zebrafish IAPs. A link
between the extrinsic and intrinsic pathways in zebrafish is considered
confirmed based on Eimon et al. [24] although it remains unclear if
bida is responsible for mediating this interaction
embryos [66]. Among the zebrafish pro-apoptotic Bcl-2
proteins, bad and bida are notably weak inducers of apop-
tosis. Interestingly, although bad does not strongly induce
apoptosis on its own, it is capable of sensitizing embryos to
c-irradiation [66]. Knockdown of bad also prevents apop-
totic cell death in the CNS of embryos that are defective for
glucose uptake due to inhibition of the slc2a1 membrane
transporter [75]. Zebrafish bida does not share conserved
synteny with mammalian Bid, leading to speculation that it
is either functionally diverged or not a true ortholog [66].
Zebrafish BH3-only proteins are capable of triggering
apoptosis in mammalian cells, confirming a high degree of
Apoptosis (2010) 15:331349
functional conservation. Transfection of COS-1 cells with
zebrafish bad induces DNA fragmentation and chromatin
condensation [76]. Zebrafish BH3 domain peptides from
bcl2l11/bim, pmaip1/noxa, and bbc3/puma induce cyto-
chrome c release in mitochondria isolated from mouse
embryonic fibroblasts (MEFs). Significantly, none of these
peptides show activity in mitochondria isolated from Bax-/-,
Bak-/- double knockout MEFs, indicating that the zebrafish
BH3 peptides are capable of interacting with endogenous
murine multidomain pro-apoptotic proteins [66].
Important regulatory sequences are conserved between
pro-apoptotic Bcl-2 proteins in zebrafish and mammals.
Mammalian Bad is regulated by phosphorylation of three
conserved serine residues, which facilitates binding of
14-3-3 proteins and promotes cell survival [77]. Two of
these residues (serines 136 and 155 in mice) are conserved
in zebrafish bad and appear to play a similar regulatory
role. When they are changed to aspartate to mimic phos-
phorylation, zebrafish bad is no longer capable of sensi-
tizing embryos to radiation-induced apoptosis. Conversely,
when they are changed to alanine to prevent phosphory-
lation, overexpression of bad alone strongly induces
apoptosis in embryos [66]. This observation indicates that
the proteins pro-apoptotic activity is normally inhibited by
serine phosphorylation during early development. Mam-
malian Bim is regulated by ERK1/2-dependent phosphor-
ylation of serine 69 (leading to proteasomal degradation)
[78, 79], AKT-dependent phosphorylation of serine 87
[80], and localization to microtubules [81]. Zebrafish
bcl2l11/bim shows conservation of the serine 69 and 87
residues, but appears to lack an obvious dynein light chain-
binding site [66]. In mammals, the BH3-only protein Bid
plays a unique role as a bridge between the extrinsic and
intrinsic apoptosis pathways [82]. While it remains
unknown if bida fulfills a similar role in zebrafish,
sequence analysis reveals a putative caspase-8 cleavage
site that would ostensibly allow for proteolytic activation
by the extrinsic apoptosis pathway [31].
Zebrafish anti-apoptotic Bcl-2 subfamily members also
show structural and functional conservation with their
mammalian counterparts. Overexpression of zebrafish bcl2,
bcl2l/bcl-xL, mcl1a and mcl1b inhibits apoptosis induced
by ectopic expression of BH3-only subfamily members and
by c-irradiation [31, 66]. Simultaneous knockdown of both
mcl1a and mcl1b significantly decreases viability in zeb-
rafish embryos, demonstrating that anti-apoptotic Bcl-2
proteins play an important role in promoting survival
during early development. In contrast, knockdown of bcl2
has no effect on embryo viability [31]. These loss-of-
function phenotypes in zebrafish are similar to those
observed in knockout mice. Mcl-1-/- mice die around
embryonic day E4.0 [83] while Bcl-2-/- mice are viable
and show no embryonic defects [84]. Several zebrafish
339
anti-apoptotic Bcl-2 subfamily members retain sufficient
conservation to function in mammalian cells. Zebrafish
bcl2l10 promotes survival following serum withdrawal in
COS-7 cells [85] and zebrafish bcl2 promotes survival
following growth factor withdrawal in murine FL5.12 cells
[86]. In the latter example, the zebrafish bcl2 protein was
shown to bind directly to human Bim, Bax, and Puma by
immunoprecipitation.
In mammals, anti-apoptotic Bcl-2 proteins associate
with pro-apoptotic family members through their BH3
domains. It is assumed that specific patterns of interaction
exist between individual Bcl-2 family members, but the
exact hierarchy of these interactions is controversial [87,
88]. Initial studies in zebrafish show that bcl2 can inhibit
apoptosis induced by overexpression of the multidomain
pro-apoptotic protein baxa and the BH3-only proteins bida,
bmf1, bmf2, pmaip1/noxa, and bbc3/puma. In contrast, bcl2
fails to inhibit baxb and only weakly inhibits bik [31]. In
GST pull-down experiments, full-length zebrafish bcl2
protein binds strongly to the BH3 domain peptide from
zebrafish bim, binds weakly to bida, bad, and bbc3/puma,
and does not bind at all to pmaip1/noxa [66]. Additional
work is required to reconcile these observations and
determine if individual Bcl-2 family members exhibit
similar protein-protein interaction patterns in zebrafish and
mammals.
The zebrafish apoptosome and effector caspases
In response to apoptotic stimuli, pro-apoptotic Bcl-2 family
members induce mitochondrial outer-membrane perme-
abilization and trigger release of pro-apoptotic factors into
the cytosol. One such factor is cytochrome c, which binds
the adaptor Apaf-1 and the initiator caspase-9 to form a
complex known as the apoptosome. Once assembled, the
apoptosome activates caspase-9, which in turn activates the
effector caspases (e.g caspase-3, -6, and -7) through pro-
teolytic processing. Activated effector caspases cleave
multiple major cellular substrates and are responsible for
amplifying pro-apoptotic signals from both intrinsic and
extrinsic pathways. In addition to cytochrome c, two other
mitochondrial factorsSmac/Diablo and the serine prote-
ase Omi/HtrA2are released following membrane per-
meabilization and indirectly promote apoptosis by
inhibiting a family of anti-apoptotic proteins known as
inhibitor of apoptosis proteins (IAPs) [89].
Although the zebrafish apoptosome has not been char-
acterized in detail, it appears highly homologous to its
mammalian counterpart. Orthologs of all major apoptosome
components have been identified in the zebrafish genome
(Table 1) [71]. Mitochondrial fragmentation is observed
in zebrafish embryos undergoing staurosporine-induced
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apoptosis, suggesting that activation of the intrinsic apop-
tosis pathway triggers disruption of the mitochondrial outer-
membrane [22]. Zebrafish caspase-9 can rapidly trigger
caspase-3 activation and apoptosis when overexpressed
in zebrafish embryos [24]. Activity assays utilizing
caspase-specific bioluminescent substrates confirm that
both c-irradiation and staurosporine cause strong activation
of caspase-9 in zebrafish [26].
Multiple effector caspases have been identified in the
zebrafish genome (Table 1) [24, 71]. Zebrafish effector
caspases cleave many of the same protein substrates known
in mammals, including PARP and 14 novel human cas-
pase-3 substrates [21, 90]. These observations suggest that
effector caspase targets are broadly conserved between fish
and mammals. There are two zebrafish orthologs of cas-
pase-3 and at least one (caspase-3a) is capable of inducing
apoptosis following overexpression in cultured cells and
zebrafish embryos [24, 25]. Recombinant caspase-3a
shows strong activity toward the mammalian caspase-3 and
-7 substrate Ac-DEVD-MCA and weak activity toward
caspase-1, -6, -8, and -9 substrates [25]. The finding that
zebrafish caspase-3a has low but significant activity
against a mammalian caspase-6-specific substrate has led
to the suggestion that it plays dual roles, partitioned
between two different effector caspases in mammals [90].
Caspase-3 enzyme activity is detected in zebrafish embryos
following overexpression of caspase-3a and treatment by
cycloheximide [21, 25]. Activation of caspase-3 in zebra-
fish following c-irradiation and overexpression of caspase-
3a, caspase-9, and pro-apoptotic Bcl-2 family members has
been confirmed by immunohistochemistry using a cross-
reactive monoclonal antibody against the cleaved, active
form of caspase-3 [24, 31]. Structural information for
caspase-3a has been obtained by X-ray crystallography,
providing a unique level of detail for this important com-
ponent of the zebrafish apoptotic machinery [91]. In
addition to the canonical effector caspases, two novel
zebrafish caspases (caspase a and caspase b) containing
N-terminal pyrin domains and sharing greatest homology
with mammalian caspase-1 have been reported to induce
apoptosis following overexpression [92]. This may in fact
be more akin to pyroposis, a novel form of proinflamma-
tory cell death mediated by caspase-1 in mammals [93].
Zebrafish IAP proteins and IAP antagonists
IAPs have been described in both invertebrates and
vertebrates and are characterized by the presence of a
C-terminal RING domain and one to three N-terminal
baculoviral IAP repeat (BIR) domains (hence the alternate
designation BIRCBIR-containing proteins). IAPs can
interact directly with caspases and other effectors of
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Apoptosis (2010) 15:331349
apoptosis to exert anti-apoptotic functions, although not all
family members are dedicated inhibitors of apoptosis [94].
Eight IAPs have been identified in humans and at least four
of these have orthologs in zebrafish (Table 1) [71, 95, 96].
The closely related mammalian c-IAP1 and c-IAP2 genes
are represented by a single ancestral birc2/ciap1 gene in
zebrafish and other lower vertebrates [96]. Zebrafish
orthologs of the IAP inhibitors Smac/Diablo and Omi/
HtrA2 have also been identified but remain to be func-
tionally characterized (Table 1).
Several recent publications show that the IAP family
plays a critical role in endothelial cell survival and vascular
homeostasis in zebrafish. A forward genetic screen for
zebrafish vascular defects identified the tomato (tom)
mutation, which produces a hemorrhagic phenotype, vas-
cular regression, and endothelial cell apoptosis [96]. The
tom mutation is located in the zebrafish birc2/ciap1 gene
and produces a nonfunctional protein product. The anti-
apoptotic function of birc2/ciap1 in endothelial cells is
dependent upon 1) binding to TNFR-associated factor 2
(TRAF2) and 2) the E3-ubiquitin ligase activity of the
RING domain. Inhibitors of caspase-8 can rescue the birc2/
ciap-/- phenotype. These observations are consistent with
mammalian c-IAPs, which interact with TNF receptors
through TRAF2 and regulate TNFa-mediated activation of
the prosurvival NF-jB pathway through polyubiquitination
of receptor-interacting protein 1 (RIP1) [2, 97101]. A
second member of the zebrafish IAP family, birc5a/survi-
vin, has also been reported to play a role in endothelial cell
survival [95, 102]. Knockdown of birc5a/survivin results in
disorganized angiogenesis, reduced vasculature, and
increased numbers of apoptotic cells. Surprisingly, knock-
down of the closely related birc5b/survivin has a much less
dramatic effect on angiogenesis although its expression
pattern during early embryogenesis is similar to that of
birc5a/survivin [102] Knockdown of either birc5a or birc5b
also triggers apoptosis in several other tissues including
hematopoietic cells and the neural tube [102, 103].
Several lines of evidence indicate that IAPs, and more
generally endothelial cell apoptosis, are important for
normal vascular development in mammals as well as zeb-
rafish [104]. Endothelial cell apoptosis has been observed
in mouse embryos beginning at E14.5 and inhibition of this
process by the anti-apoptotic Bcl-2 protein results in
abnormally dense and disorganized networks of small
vessels [105]. Survivin-/- knockout mice die by E3.5 [106],
but selective inactivation of Survivin in endothelial cells
using the cre-lox system results in embryos that survive to
E12.5 and exhibit extensive hemorrhaging throughout the
body, diffuse vascular endothelial apoptosis, and increased
pericyte-endothelial gaps [107]. Although there are no
reports of vascular defects in mice deficient for c-IAP1 or
c-IAP2 [108, 109], this may be due to the high degree of
Apoptosis (2010) 15:331349
conservation between these two family members, which
allows for functional redundancy in some contexts [99,
100]. This hypothesis is supported by the finding that
simultaneous depletion of c-IAP1 and c-IAP2 in human
umbilical cord vascular endothelial cells (HUVECs) sen-
sitizes these cells to killing by TNFa, the TNFR1 ligand
[96].
Zebrafish p53 and the intrinsic apoptosis pathway
The tumor suppressor protein p53 is a multifunctional
transcription factor that plays a central role in determining
how cells respond to stresses such as DNA damage,
hypoxia, and oncogenic signaling [110]. In stressed cells,
p53 is stabilized and converted from a latent to an active
form. Activation of p53 can lead to a variety of outcomes,
including cell cycle arrest (allowing for DNA repair) and
apoptosis (to eliminate cells damaged beyond repair). In
the latter case, p53-dependent apoptosis is primarily med-
iated by stimulation of the intrinsic apoptosis pathway.
Stabilized p53 accumulates in the nucleus and can directly
regulate the expression of many pro-apoptotic genes (e.g.
Bid, Noxa, Puma, Apaf1). Mounting evidence indicates
that transcription-independent activities also play an
important role in p53-induced apoptosis. Specifically, p53
can translocate to the mitochondrial outer membrane fol-
lowing induction and directly interact with both pro- and
anti-apoptotic members of the Bcl-2 family [111, 112].
The tp53 gene is a critical mediator of apoptosis in
response to DNA damage in zebrafish. A variety of DNA
damaging reagents have been shown to upregulate zebra-
fish tp53 transcription [29, 113] and increase tp53 protein
levels [114]. Knockdown of tp53 blocks apoptosis arising
from a wide range of stressors, including c-irradiation [32],
ultraviolet irradiation, camptothecin treatment [29], inhi-
bition of the DNA crosslink repair gene fancd2 [115], and
compromised DNA replication in mutant lines such as
flathead, caf1b, and pinball eye [116118]. A similar
degree of protection is observed when embryos are pre-
treated with Pifithrin-a, a pharmacological inhibitor of p53
[119]. Resistance to DNA damage-induced apoptosis is
also seen in tp53M214K (sometimes referred to as tp53e7), a
zebrafish line with a dominant negative mutation in the
tp53 locus that destroys transcriptional activity [113]. In
addition to reduced apoptosis, the tp53M214K line begins to
develop malignant peripheral nerve sheath tumors at
approximately 8.5 months of age, confirming tp53 is a
critical tumor suppressor protein in fish. The related tran-
scription factors tp63 and tp73 do not appear to play sig-
nificant roles in apoptosis in zebrafish [120, 121].
As in mammals, zebrafish tp53 autoregulates its own
expression [29] and promotes transcription of a diverse
array of target genes, including cdkn1a/p21 [29], mdm2 and
341
baxa [113], perp [122], and bbc3/puma [31]. Puma has
been identified as a key mediator of p53-dependent apop-
tosis in mammals based on data from knockout mice [123]
and bbc3/puma appears to play a similarly essential role in
zebrafish [31]. Upstream regulation of tp53 has also been
characterized in zebafish. As in mammals, the E3 ubiquitin
ligase mdm2 serves as a primary negative regulator of tp53
activation. Knockdown of zebrafish mdm2 results in
extensive apoptosis, which can be rescued by simultaneous
knockdown of tp53 [29]. Other ubiquitin ligases known to
negatively regulate p53 in mammalssuch as COP1 [124]
and Pirh2 [125]are present in the zebrafish genome but
have not been functionally characterized. Epistasis analysis
in piy mutant embryos shows that two genes known to
mediate the DNA damage response upstream of p53 in
mammalsataxia telangiectasia mutated (atm) and
checkpoint kinase 2 (chek2)are also essential for tp53-
dependent apoptosis in zebrafish [116]. Seven novel acti-
vators of p53 (Hey1, Hes1, TFAP4, Osr1, NR2F2, SFRS10,
and FLJ11339), initially identified in a high-throughput
in vitro screen using a human cell line, are capable of
inducing tp53 expression in zebrafish embryos [126].
Several recent studies have used zebrafish to identify and
validate novel mechanisms of p53 regulation that are
broadly conserved throughout vertebrate evolution. Le et al.
[127] identified miR-125b, a brain-enriched microRNA
(miRNA), as a potential negative regulator of p53, by
searching for miRNA response elements that have been
conserved between zebrafish and humans. Overexpression
of miR-125b reduces p53 levels and inhibits p53-dependent
apoptosis in primary human lung fibroblasts and the neu-
roblastoma cell line SHSY5Y. In zebrafish, knockdown of
miR-125b leads to increased apoptosis in wild-type
embryos, but not in embryos from the tp53M214K mutant
line. Injection of synthetic miR-125b duplex into zebrafish
embryos rescues the knockdown phenotype and inhibits
apoptosis following DNA damage. Chen et al. [128] show
that D113p53, the zebrafish counterpart of the human p53
isoform D133p53, is activated in a p53-dependent manner
following DNA damage and functions to antagonize
apoptosis, in part through upregulation of bcl2l/bcl-xL.
Knockdown of endogenous D113p53, using a morpholino
targeting the D113p53 5-UTR, increases the sensitivity of
embryos to c-irradiation-induced apoptosis.
Sidi et al. [86] have used the zebrafish tp53M214K mutant
to better understand p53-independent apoptotic responses
and gain important insights into the function of caspase-2.
Knockdown of checkpoint kinase 1 (chek1) was found to
restore sensitivity to c-irradiation-induced apoptosis in
tp53M214K embryos. Surprisingly, although dying cells in
chek1-knockdown embryos are TUNEL positive, caspase-3
activation is not observed and cell death cannot be blocked
by bcl2l/bcl-xL overexpression or bbc3/puma knockdown.
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Epistasis analysis demonstrates that zebrafish orthologs
of ATM, ATR, and the initiator caspase-2 are critical for
radiation-induced apoptosis in tp53M214K/chek1-knock-
down embryos. Experiments in HeLa cells suggest that a
similar Chek1-suppressed caspase-2 pathway exists in
mammalian cells defective for p53 activity.
ATM and ATR are well-characterized apical kinases
that respond to DNA damage by activating a number of
downstream proteins including p53 [129]. The precise role
of caspase-2 in apoptosis, however, is a matter of long-
standing debate [3]. In mammalian systems caspase-2 has
been linked to both the intrinsic and extrinsic pathways,
although it does not appear to be required for cell death in
most contexts [130]. The mechanism of caspase-2 acti-
vation has not been fully elucidated, although it is thought
to involve a protein complex similar to the apoptosome
that incorporates the adaptor proteins RAIDD and PIDD
(designated the PIDDosome) [131]. Orthologs of both
RAIDD (cradd) and PIDD (LOC571033) have been
identified in the zebrafish genome (Table 1) and overex-
pression of casp2 on its own is sufficient to induce
apoptosis in embryos [24]. The roles of the RAIDD and
PIDD orthologs in zebrafish have yet to be studied and
their requirement in the novel Chek1-suppressed caspase-2
pathway is unclear.
Fig. 2 Zebrafish Death Receptors and Ligands. The diagram illus-
trates all TNF and TNFR superfamily members that have been
reported to date in zebrafish. Official zebrafish gene designations are
given in lower case italic type and corresponding mammalian
homologs are given in upper case bold. Red arrows indicate ligand-
receptor interactions that have been confirmed in zebrafish by
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Apoptosis (2010) 15:331349
The cell-extrinsic apoptosis pathway
Zebrafish death receptors and ligands
The extrinsic apoptosis pathway is engaged when certain
ligands of the TNF superfamily bind to specialized mem-
bers of the TNFR superfamily known as death receptors.
Death receptors are characterized by the presence of a
conserved *80-amino-acid death domain motif in the
cytoplasmic tail that plays a central role in formation of the
DISC [132]. The human genome contains six death
receptors and five cognate TNF ligands (Fig. 2). An
extracellular fragment of b-amyloid precursor protein
(APP) has recently been shown to act as a ligand for DR6
and drive axonal degeneration and neuronal death in mice
[133]. This arrangement of receptors and ligands is well
conserved in mammals, although many species possess
only a single ortholog of the closely related death receptors
designated DR4 and DR5 in humans and other primates
[134].
Recent studies have shed considerable light on death
receptor ligands in zebrafish and other teleosts. Orthologs
of TNF (tnfa and tnfb), CD95/FasL (faslg), Apo2L/TRAIL
(tnfsf10l, tnfsf10l2, tnfsf10l3, and tnfsf10l4), and APP
(appa) have been identified based on phylogenetic analysis
co-immunoprecipitation studies and/or antisense morpholino oligo-
nucleotide knockdown experiments. Gray arrows indicate interac-
tions that are hypothesized to occur based on studies in other
vertebrate species but remain to be experimentally verified in
zebrafish. The zebrafish homolog of APP is included as a potential
ligand for tnfrsf21/dr6 based on recent evidence in mammals
Apoptosis (2010) 15:331349
[24, 135139]. Co-immunoprecipitation studies confirm
that tnfa binds selectively to tnfrsf1a, the zebrafish ortholog
of TNFR1, as expected [24]. A probable zebrafish TL1A
ortholog (LOC560966) has also been described based on
phylogenetic analysis and the presence of conserved cys-
teine residues [135]. The status of LTa in zebrafish and
other teleosts remains unclear. Savan et al. [137] describe a
novel TNF gene (TNF-N; subsequently designated lta) that
appears similar to mammalian LTa based on its proximity
to the zebrafish tnf locus. However, the intronic structure of
lta is distinct from human LTa and it clusters with OX40L,
GITRL, and CD30L in an unrooted phylogenetic tree
[135].
Of the four zebrafish Apo2L/TRAIL homologs,
tnfsf10l2 exhibits the greatest structural similarity with
mammalian Apo2L/TRAIL [24]. Both tnfsf10l and
tnfsf10l3 are more structurally related to Apo2L-likea
second copy of Apo2L/TRAIL found in avian species but
not mammalsdue to additional cysteine residues in their
extracellular domains [140]. Zebrafish tnfsf10l4 shows the
greatest sequence divergence and only groups weakly with
other Apo2L/TRAIL and RANKL sequences [135].
Death receptors are also well conserved between zeb-
rafish and mammals. Clear orthologs of TNFR1 (tnfrsf1a),
CD95/Fas (fas), and DR6 (tnfrsf21) have been identified
based on the arrangement of their cysteine-rich extracel-
lular domains, phylogenetic analysis of their death
domains, and conserved synteny with mammalian genes
(Table 1) [24]. In addition, two death domain-containing
receptors designated hdr [141, 142] and tnfrsfa (previously
known as Ovarian TNF Receptor or otr) [139] have been
described in zebrafish. These receptors have less clear-cut
mammalian orthologs. They contain extracellular domains
that are most similar to CD95/Fas and death domains that
are most similar to DR4 and DR5. The finding that both hdr
and tnfrsfa bind Apo2L/TRAIL orthologs and are essential
for Apo2L/TRAIL-induced apoptosis in zebrafish argues
strongly for categorizing them as homologs of DR4 and
DR5 (Fig. 2) [24]. DR3 remains the only mammalian death
receptor without a zebrafish counterpart, although the
identification of a zebrafish ligand with homology to TL1A
[135] suggests such an ortholog may yet be found.
Components of the zebrafish DISC
Activated death receptors mediate downstream activity
through one of two distinct signaling complexes. Engage-
ment of the adaptor protein FADD leads to DISC recruit-
ment and drives proapoptotic signaling through caspase-8
(as well as caspase-10 in human cells) [132]. Activation of
caspase-8 is positively controlled by Cullin-3-mediated
polyubiquitination and p62-dependent aggregation [143]
and negatively controlled by the deubiquitinase A20 [143]
343
and cellular FLICE-inhibitory protein (c-FLIP), an enzy-
matically inactive relative of caspase-8 and -10 [144]. In
contrast, engagement of the adaptor protein TRADD leads
to assembly of Complex I, which induces transcription of
both proinflammatory cytokines and antiapoptotic factors
through the IKK/NF-jB, JNK, and p38 MAPK pathways
[145]. In mammals, only DR4, DR5, and CD95/Fas
which signal primarily through FADDare direct activa-
tors of the extrinsic apoptosis pathway. TNFR1 and DR3
bind TRADD and mediate immune-stimulatory activity.
Cross talk is possible between these two distinct death
receptor signaling pathways through distal signaling com-
plexes that are nucleated by FADD and TRADD [2].
Zebrafish orthologs have been identified for most com-
ponents of the DISC and Complex I, including the pivotal
adaptor proteins FADD and TRADD (Table 1) [24, 71].
Reverse genetic analysis has been used to interrogate the
requirement for fadd and tradd in the zebrafish DISC.
Inhibition of fadd, but not tradd, is sufficient to block
apoptosis induced by overexpression of the Apo2L/TRAIL
orthologs tnfsf10l and tnfsf10l3 in zebrafish embryos [24].
Transient overexpression of zebrafish death receptors
(tnfrsf1a, fas, hdr, and tnfrsfa) and fadd in human cells
triggers apoptosis, indicating that the death domain motif
has been sufficiently well conserved between teleosts and
mammals to enable cross-species protein interactions
within the DISC [24].
A single zebrafish ortholog of caspase-8 (casp8a), con-
taining N-terminal death effector domains (DEDs) and a
QACQG active-site motif characteristic of mammalian
caspase-8 and -10, has been described [24, 146]. Several
lines of evidence show that zebrafish casp8a is functionally
conserved with its mammalian counterpart. FADD directly
associates with casp8a through homophilic DED interac-
tions [146]. Zebrafish casp8a can mediate apoptosis fol-
lowing death receptor activation in mouse embryonic
fibroblast (MEF) cells that lack endogenous caspase-8
[146]. The cowpox virus serpin cytokine response modifier
A (CrmA)which inhibits caspase-8, -10, and -1 in
mammalsblocks Apo2L/TRAIL-induced apoptosis in
zebrafish embryos [24].
Additional caspase-8 related genes are present in zeb-
rafish (Table 1) [24, 146]. These include casp8l1, casp8l2,
and three genes containing an N-terminal caspase recruit-
ment domain (CARD) in place of the DED motifs (caspxa,
LOC795066, and zgc:194469). Casp8l1 contains the dis-
tinctive QACQG active-site motif but lacks N-terminal
DEDs. In contrast, casp8l2 possesses DEDs but contains a
QACRG active site that is more reminiscent of caspase-2.
In addition, zebrafish possess a clear ortholog of c-FLIP
(cflar) that contains N-terminal DEDs and an enzymati-
cally inactive caspase cataltic domain. Overexpression of
cflar in zebrafish embryos prevents induction of apoptosis
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by the Apo2L/TRAIL orthologs tnfsf10l and tnfsf10l3 [24],
consistent with its roles as an inhibitor of death receptor-
mediated apoptosis in mammals.
Biological roles of the extrinsic apoptosis pathway
in zebrafish
Although the extrinsic pathway is critical for normal
functioning of the mammalian immune system, it remains
unclear if it plays a similar role in zebrafish. In humans,
mutations in CD95/Fas, CD95L/FasL, and caspase-10 are
responsible for autoimmune lymphoproliferative syn-
drome, while mutations in caspase-8 cause deficiencies in
both lymphocyte proliferation and activation [2, 147].
Knockout studies in mice confirm that the extrinsic path-
way has widely conserved roles in immune system regu-
lation [3, 148, 149]. In zebrafish, T and B cells do not
develop until 3 dpf or later [150], making analysis of the
extrinsic pathway in these cell types by morpholino
knockdown techniques impractical. Langenau et al. [151]
have used a stable transgenic line expressing bcl2 under the
control of the rag2 promoter to show that apoptosis plays
an integral role in zebrafish T- and B-cell homeostasis, but
additional transgenic or mutant lines will be required to
verify a specific role for the extrinsic pathway in these cell
types. Two studies demonstrate that the zebrafish death
receptor hdr plays a homeostatic role in at least one non-
lymphoid cell type. Long et al. [141] found that hdr is
specifically expressed in the hematopoietic lineage and
used transgenic zebrafish expressing a dominant negative
hdr version to demonstrate that receptor inhibition leads to
an accumulation of red blood cells. Kwan et al. [142]
reported similar findings in zebrafish embryos following
morpholino knockdown of hdr. In the context of these
observations, it is interesting to note that overexpression of
tnfsf10l3, the hdr ligand, selectively induces apoptosis in
the erythroid lineage [24].
Embryonic gene expression patterns suggest that death
receptor signaling may play a role in numerous zebrafish
tissues [24]. In addition to the hematopoietic lineage, hdr is
strongly expressed in the developing notochord during
early development. The closely related death receptor
tnfrsfa is also expressed in the notochord during this per-
iod, as is fadd. Tnfrsfa and the Apo2L/TRAIL homologs
tnfsf10l2 and tnfsf10l3 are detected in neuromasts of the
lateral line. These mechanosensory organs contain hair
cells similar to those of the mammalian inner ear and are
known to undergo constant, apoptosis-mediated turnover in
zebrafish [152]. Finally, several death receptors (tnfrsf1a,
tnfrsfa, and tnfrsf21/dr6) and ligands (tnfsf10l, tnfsf10l2,
and tnfsf10l3) are expressed in regions of the developing
CNS. Their role in zebrafish remains unknown, but in
mammals the expression of death receptors and ligands
123
Apoptosis (2010) 15:331349
(particularly CD95/Fas and DR6) in the CNS is thought
to influence a variety of neuropathological processes
[133, 153].
Concluding remarks
Invertebrate model organisms such as C. elegans and
Drosophila have been used widely and successfully to
study apoptosis [154, 155]. The ease with which these
systems can be genetically manipulated has been instru-
mental in identifying and characterizing core components
of the apoptotic machinery and understanding how these
molecules are regulated under both normal and pathologi-
cal conditions. In spite of their advantages, invertebrate
models cannot replicate many aspects of apoptosis known
to be important in mammals. Apoptosis that occurs as a
normal part of embryogenesis and adult tissue homeostasis
is often specific to organs and tissues that lack analogs in
invertebrate species. In other cases, apoptosis arises from
disease pathologies that cannot be modeled in nematodes
and flies. Additionally, apoptotic pathways have evolved to
a far greater degree of complexity and diversity in mam-
mals and other vertebrates (Fig. 1) [156, 157]. For exam-
ple, more than 25 Bcl-2 superfamily members have been
identified in humans [62] versus three in C. elegans (egl-1,
ced-9, and ced-13) [158] and two in Drosophila (buffy and
debcl) [159]. An even more extreme case is the TNFR
superfamily, comprising approximately 30 members in
humans including six death domain-containing receptors
responsible for engaging the extrinsic pathway [160]. This
superfamily is not present at all in C. elegans and consists
of only a single member lacking a death domain (eiger) in
Drosophila [161]. Finally, there are fundamental differ-
ences in the way apoptotic pathways function between
invertebrates and mammals. Drosophila Bcl-2 proteins
appear to be peripheral players responsible for fine-tuning
stress-induced apoptosis [162, 163], while in mammals
they serve as the chief regulators of the intrinsic pathway.
This difference may be linked to the increased importance
of mitochondria as activators of the intrinsic pathway in
mammals versus C. elegans and Drosophila. In mammals,
Bcl-2 proteins regulate the release of pro-apoptotic mole-
cules such as cytochrome c, Smac/Diablo, and Omi/HtrA2
from the mitochondria. Unlike mammals, cytochrome c
does not play a role in apoptosome formation in C. elegans
and its role in Drosophila appears to be extremely limited
[159, 164]. The Reaper, Grim, and Hid (RGH) proteins
functional counterparts of Smac/Diabloplay a central
role in Drosophila apoptosis but are regulated primarily via
increased expression rather than mitochondrial sequestra-
tion [162, 165]. Fundamental differences are also apparent
in the extrinsic pathway. Eiger triggers cell death through a
Apoptosis (2010) 15:331349
JNK-dependent mechanism using the adaptor protein
TNF-receptor-associated factor 6 (TRAF6, formerly known
as dTRAF2) [161, 166]. Drosophila homologs of FADD
(dFADD) and caspase-8 (Dredd), have not been linked to
the extrinsic pathway and instead appear to promote tran-
scription of antimicrobial peptide genes following bacterial
infection [167].
Due to these limitations, a vertebrate model of apoptosis
that is genetically tractable, amenable to analysis during
early development, and suitable for experimental approa-
ches with relatively high throughput is desirable. The
zebrafish fulfills all of these criteria and, as this review
shows, has a diversity of apoptosis-related genes compa-
rable to mammals (Fig. 1). In addition to preserving the
complexity of mammalian systems, zebrafish apoptosis
pathways are functionally more analogous to mammals
than invertebrates on a number of levels. The Bcl-2 family
appears to play a far more crucial role in regulating the
intrinsic pathway in zebrafish than Drosophila, as demon-
strated by the rapid and extensive cell death triggered by
knockdown of mcl1a and mcl1b, the requirement for bbc3/
puma in DNA damage-induced apoptosis, and the ability of
anti-apoptotic family members to inhibit apoptosis in
response to both stress stimuli and extrinsic pathway acti-
vation [24, 31, 151]. In contrast to Drosophila, the zebra-
fish extrinsic pathway requires both FADD and caspase-8
to trigger apoptosis and undergoes amplification through
the intrinsic pathway, similar to type II cells in mammals
[24]. These findings suggest the zebrafish has the potential
to become a powerful in vivo model for studying cell death.
It remains important to verify a number of features of
zebrafish apoptosis. In particular, the roles of the mito-
chondria and mitochondrial factors such as cytochrome c,
Smac/Diablo, and Omi/HtrA2 need to be clarified, as these
are among the most striking points of divergence between
invertebrates and mammals. Demonstrating an essential
role for cytochrome c (zgc:86706) in formation of the
zebrafish apoptosome may require a three-dimensional
structure of the complex, but it should be possible to
address changes in the subcellular localization of Smac/
Diablo and Omi/HtrA2 homologs with far less difficulty.
Fish models may provide unique advantages for the
study of apoptosis [168, 169]. Zebrafish and other teleost
species are useful for studying apoptotic cell death occur-
ring not only during embryonic development but also in
response to a variety of external stimuli including changes
in oxygen availability, osmotic stress, high and low tem-
peratures, and exposure to toxins. Many small molecule
drugs exhibit conserved activities between zebrafish and
humans [170, 171], raising the possibility that zebrafish
assays may also prove useful for identifying novel drugs
designed to induce apoptosis for the treatment of cancer.
For example, the highly specific phenotypes observed in
345
zebrafish embryos following inhibition of IAPs [96] or
ectopic expression of Apo2L/TRAIL homologs [24] could
serve as the basis for in vivo screens to identify small
molecules with similar pro-apoptotic activities. It is likely
that future apoptosis research in zebrafish will continue to
exploit the strengths of this model system, in particular its
genetic tractability. To date, loss-of-function mutants have
been reported only for a handful of the apoptosis-related
genes listed in Table 1. Therefore, it will be important to
identify and characterize additional mutant lines that are
deficient in core components of the intrinsic and extrinsic
apoptosis pathways. The failure of large-scale forward
genetic screens to identify such mutants so far suggests that
reverse genetic techniques, such at TILLING and gene
targeting through zinc-finger nucleases, may be the pre-
ferred approaches. The value of such genetic tools is nicely
demonstrated by recent publications using birc2/ciap1 and
tp53 mutant lines to elucidate novel apoptosis pathways,
regulatory mechanisms, and physiological functions
[86, 96, 127, 128].
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