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Apoptosis (2010) 15:331–349

DOI 10.1007/s10495-009-0432-9


The zebrafish as a model organism for the study of apoptosis
Peter M. Eimon • Avi Ashkenazi

Published online: 24 December 2009
Ó Springer Science+Business Media, LLC 2009

Abstract Apoptosis plays important roles in embryo- Introduction
genesis, tissue homeostasis, and immune system regula-
tion. The zebrafish (Danio rerio) is a powerful vertebrate There are two key apoptotic signaling mechanisms in
model organism that has been extensively used to study mammals and other vertebrates: the cell-intrinsic (or
apoptotic cell death during normal development and under mitochondrial) pathway and the cell-extrinsic (or death
conditions of cellular stress. In the past 5 years, a detailed receptor) pathway. The intrinsic pathway can be triggered
picture has begun to emerge of the molecular underpin- by a variety of cues including cellular damage, growth
nings of the cell-intrinsic and the cell-extrinsic apoptosis factor withdrawal, and chemotherapeutic drugs. It is reg-
signaling pathways in zebrafish. We begin this review with ulated by the Bcl-2 gene family and functions through the
an introduction to the techniques and experimental initiator protease caspase-9 [1]. The extrinsic pathway
approaches that are used to study apoptosis in zebrafish. plays important roles in immune system operation and
We follow with a general overview of developmental homeostasis. It is triggered by death receptor ligation and
apoptosis during zebrafish embryogenesis. Finally, we functions through a death-inducing signaling complex
present a comprehensive review of the intrinsic and (DISC) that includes the initiator proteases caspase-8 and
extrinsic apoptosis pathways in zebrafish, focusing on the -10 [2]. The extrinsic pathway is linked to the intrinsic
high degree of conservation with humans and other mam- pathway through Bid, a Bcl-2 family protein that can be
mals. Recent publications that draw upon the unique cleaved and activated by caspase-8. Additional pathways,
advantages of the zebrafish system to study novel aspects such as the granzyme B and endoplasmic reticulum path-
of apoptosis regulation and function are highlighted ways, may also mediate aspects of apoptosis under certain
throughout. conditions. All pathways converge at the level of the
effector caspases (caspase-3, -6 and -7), which carry out
Keywords Apoptosis  Zebrafish  Intrinsic pathway  the molecular mechanics of programmed cell death [3].
Extrinsic pathway  Bcl-2  IAP  Death receptor  The intrinsic apoptosis pathway appears to have evolved
Apo2L/TRAIL  Caspase 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-
P. M. Eimon gans). Death receptors (TNFRs possessing a cytoplasmic
Zygogen, LLC, Atlanta, GA, USA death domain motif) have been reported almost exclusively
in vertebrates, although recently a death domain-containing
A. Ashkenazi (&)
TNFR has been identified in the Zhikong scallop (Chlamys
Department of Molecular Oncology, Genentech Inc.,
1 DNA Way, MS 42, South San Francisco, CA 94080, USA farreri) [4]. In the following review we will summarize the
e-mail: current understanding of the intrinsic and extrinsic


brate biology. cleaved. LocusLink (LOC) designations are used for Several experimental protocols have been developed zebrafish genes without an official full name. undergo rapid and synchronous devel. These include their small size (embryos as this are crucial. and zinc. pair can produce hundreds of embryos per week). The hallmarks between fish and mammals. 8]. and are optically transparent. and neuromasts [19]. 26].org/Danio_rerio/Info/Index). official zebrafish gene names are model organism as highly relevant for the study of verte- paired with their more familiar human designation (e. More com- larities and differences between these pathways in zebra.332 Apoptosis (2010) 15:331–349 apoptotic pathways in zebrafish. mammals. acteristic of apoptotic cells [18. apoptotic cells in vivo [22]. and genetic tractability. Zebrafish have served as an important non-specific uptake in the yolk. that allow apoptotic cells to be visualized in vivo in zeb- rafish embryos. Finally. hatching research organism for decades.sanger.7 mm in diameter at fertilization and grow to embryo-to-embryo variability and large numbers of 3. of periderm cells [19]. a transgenic zebrafish line expressing mitochond- active form of caspase-3 [24]. iridophores. high fecundity (a single zebrafish embryos must be analyzed to ensure statistical rigor. lowing several publications in 1996 demonstrating that Many assays used to detect apoptosis in mammalian they are amenable to large-scale forward genetic screens tissue samples and cell culture have been adapted to fixed and describing over 1000 mutants with visible phenotypes zebrafish embryos and embryonic lysates. short Hoechst 33342. A unique combi. tilized externally. elegans and Drosophila melanogaster.5 mm within 3 days). Techniques have been activity of caspase-3. a nucleic Approaches to studying apoptosis in zebrafish acid intercalating dye that emits green fluorescence when bound to dsDNA and selectively stains apoptotic rather The zebrafish is a small freshwater teleost of the cyprinid than necrotic cells [17. (http://www. Approaches such vertebrate model. tar. has also been generation time (adults reach sexual maturity used in live zebrafish embryos to visualize the formation of in *3 months). Analysis of AO staining in family (carps and minnows) in the class of ray-finned fishes zebrafish embryos is complicated by autofluorescence or (Actinopterygii). Fixed embryos during early development [7. According to some Many additional tools further enhance the utility of reports. and protocols have been developed rafish embryos and report broad conservation of apoptotic for proteomic and metabolomic analysis [15. which detects fragmented DNA char- such as C. an exceptionally gene names and symbols endorsed by Entrez and the large number of genes and molecular pathways have been Zebrafish Information Network (ZFIN) throughout this well characterized in zebrafish and help to validate this review. additional techniques to characterize the biochemical Commercial zebrafish DNA microarrays are available for modifications associated with apoptotic cell death in zeb- expression profiling. and caspase-9 in zebrafish devised for generating stable transgenic lines that express embryo lysates and in dissociated cells [21.ensembl. Embryos are fer. 18]. a DNA-binding vital dye. we have used the official As a result of these and other approaches. tency in zebrafish nomenclature. chromatin clumps characteristic of apoptotic nuclei [21]. We will highlight simi. Previously. In some cases. A recent publication describes genetics and developmental biology [5]. 16]. Fluorogenic and finger nucleases to generate locus-specific mutations for bioluminescent substrates have been used to monitor the targeted gene inactivation [11]. use of the TUNEL method on zebrafish embryos zebrafish as a model organism. fluorescent reporter proteins or other genes of interest Negron and Lockshin [21] have used a number of under the control of tissue-specific promoters [12–14]. and more divergent model organisms such tion with ZFIN and can be accessed through the Vega as fruit flies and nematodes.g. Extensive intranu- Sanger Institute has sequenced the zebrafish genome cleosomal DNA fragmentation was confirmed by agarose and an annotated assembly is available in Ensembl gel electrophoresis and poly(ADP-ribose) polymerase 123 .html). tnfrsf21/dr6). Fixed embryos can also be immu- geting induced local lesions in genomes (TILLING) to stained using a monoclonal antibody that recognizes the identify stable mutations in genes of interest [10]. since AO staining exhibits considerable are *0. large-scale can be stained by the terminal dUTP nick end-labeling screening approaches had been limited to invertebrates (TUNEL) method. Reverse genetic approaches older than 48 h post fertilization (hpf) is hampered by include using antisense morpholino oligonucleotides to unacceptable background staining and nonspecific labeling knock down gene function transiently in embryos [9]. 25. developed to monitor mitochondrial fragmentation in The popularity of zebrafish increased substantially fol. database (http://vega. caspase-8. particularly in the fields of gland. Most organs become rially-targeted green fluorescent protein (GFP) has been functional between 3 and 5 days post fertilization (dpf) [6]. an imaging protocol that enables rapid quantification of AO nation of features makes them particularly attractive as a staining in large groups of embryos [20]. 23]. One of the most commonly used tech- niques utilizes the vital dye acridine orange (AO). In order to promote consis. plete manual annotation is being carried out in collabora-

Rohon- cell death is truly apoptotic. Competition between neurons for limiting amounts of muscle-derived neurotrophic factors is thought to be a In multicellular organisms. A similar inhibition of phase peaking at 3–4 dpf and a second phase at 6–7 dpf 123 . like mammals. Additional forms of programmed cell death— ment. occurring cell transplant and cell ablation studies. seen in the developing zebrafish retina. and cell migration. ultra. a second motoneuron (designated VaP) is particularly autophagy (or type II cell death)—are utilized often present alongside CaP in the spinal cord. In contrast. In common mechanism employed during development to adult zebrafish. one well-characterized period of apoptosis occurs when axons contact their postsynaptic Developmental apoptosis in zebrafish targets. cycloheximide [21]. eyes (peak- necrosis share a number of common features and it is ing at 36 hpf in the retina and 20–48 hpf in the lens). Chemical (peaking between 30–36 hpf). stress-induced apoptosis in zebrafish can be widespread in the central and peripheral nervous systems blocked by the pan-caspase inhibitor Z-VAD-FMK and by of most vertebrates during development. Cell death is mammals. In zebrafish. Apoptosis is the most regulation of motoneuron apoptosis in greater detail. the cytoprotective agent amifostine [26]. Apoptotic the apoptotic machinery in pre-gastrula stage embryos has cells translocated phosphatidylserine—a component of the been documented in Xenopus laevis [37]. 38]. prior to the end of MZT exhibit an arrest of cell prolifer. and death. as it is not impacted by contin. and it Zebrafish embryos are resistant to apoptosis before they undergoes apoptotic death by 36 hpf. 35]. with an initial uous treatment with cycloheximide. Similar patterns of developmental apoptosis have camptothecin [28. differentiation. however. During early develop- [33. Transmission electron micros. Similar to several zebrafish neuronal populations. In many regions. A similar pattern of apoptosis is post-translational control. Although over-production of cells that will axons that pause briefly at an intermediate target known as ultimately be eliminated by apoptosis may appear ener. trigeminal Many experimental methods for inducing apoptosis in neurons (peaking between 36–48 hpf). Both CaP in certain developmental contexts but fall outside the scope and VaP are morphologically equivalent and send out of this review. The cell whose axon first getically wasteful. In motoneurons. apoptosis and including the olfactory placodes (22–72 hpf). Cole nuclear membrane. Beard neurons (peaking between 36–48 hpf). 36]. where two consecutive phases of apoptosis have been ation but survive until the mid-gastrula stage (*7 hpf). 44]. or otherwise unwanted cells single motoneuron designated CaP. the authors show between 4 and 7 hpf). Through a series of reach the maternal-to-zygotic transition (MZT. or staurosporine widespread among neurons of the mammalian retina. innervate the ventral myotome.Apoptosis (2010) 15:331–349 333 (PARP) cleavage was detected by Western blot. Engulfment of apoptotic cell corpses by and Ross [39] have used the TUNEL method to map neighboring cells has also been observed in zebrafish developmental apoptosis in zebrafish between gastrulation embryos using electron microscopy. at described. cycloheximide. apoptotic cells are detected in exhibit chromatin condensation and convolution of the various organs and tissues of zebrafish embryos [19]. nocodazole. 28. normal development depends major driver of apoptosis in this context [42]. more than 50% of newly formed neurons are eliminated [41]. hydroxyurea been observed in other fish species such as medaka [28]. while the later phase occurs during Acquisition of apoptotic competence appears to be under target innervation [45. been demonstrated in response to heat shock [30]. 46]. it is a common feature of metazoan reaches the HM adopts the CaP fate and goes on to development and occurs in species ranging from nema. and c-irradiation [30–32]. The remaining cell adopts todes to humans [34. misplaced. apoptotic cell-surface ‘‘eat-me’’ signal—to the outer leaflet apoptotic cell death has been reported in mammalian of the plasma membrane. 29 hpf). embryos as early as the 16-cell stage [34. and tail bud (peaking at reagents that have been evaluated include aphidicolin [28]. Eisen and upon a carefully orchestrated balance between cell prolif. the stage during early development that VaP motoneuron death is governed by a death-pro- when the zygotic genome becomes the primary source of moting signal originating from HM cells and triggered by mRNA in the embryo [21. Embryos treated with the CaP axon [43. Developmental apoptosis is also camptothecin. copy was used to confirm that apoptotic cells in zebrafish Following gastrulation. 29]. the horizontal myoseptum (HM). The authors describe transient high levels of inhibited by knockdown of the phosphatidylserine receptor developmental apoptosis in a number of structures jmjd6 [27]. Developmental apoptosis has been studied in detail in violet irradiation [29]. each ventral myotome is innervated by a remove damaged. lat- important to use two or more distinct assays to confirm that eral line neuromasts (beginning at 24–30 hpf). Apoptotic cell death has also (Oryzias latipes) [40]. its axon remains paused at the HM. Melanc¸on [43] have used zebrafish embryos to study the eration. urogenital system mammalian cells are also effective in zebrafish. engulfment can be and 4 dpf. The earlier phase occurs during neurogenesis which point they undergo rapid and synchronous cell death. 34]. and staurosporine [22]. the VaP fate.

BH3. Unfortunately. The inner ear is another region where the role of (designated BH1. but rather serves to eliminate cells that are also been characterized in zebrafish. remainder of this review will focus specifically on the In some cases. similar to a loss-of-function phenotypes characterized by excessive mechanism reported during development of the rat lens apoptotic cell death (reviewed in Pyati et al.g. many hallmarks of apoptosis including chromatin frag. of mammals. the total number of apoptotic cells detected in [60]). while the second ples. molecular mechanism governing the intrinsic and extrinsic cesses that have not been evolutionarily conserved between apoptosis pathways in zebrafish. or otherwise deficient due to dis- apoptotic cells are detected in the zebrafish lens around ruptions in the tightly regulated process of embryogenesis. By far the formed by the BH1. Interactions Large-scale mutagenesis screens in zebrafish have between subfamily members occur when the BH3 domains identified numerous genes with loss-of-function pheno. Anti-apoptotic developmental apoptosis has not been conserved through. and BH3 domains on anti- largest class of these mutants is associated with elevated apoptotic proteins [65]. Bik.g. or suppression of the anti-apoptotic subfamily. BH2. for exam- (GCL) and the inner nuclear layer (INL). Members of the BH3- Xenopus laevis failed to detect a similar hot spot.334 Apoptosis (2010) 15:331–349 [47]. For a more may also arise from programmed removal of nuclei and detailed discussion of mutants and morpholino knockdown other organelles from lens fiber cells. These primary sensory neurons are found in anamniote verte. BH2. A thorough examination of apop. and the Bnip proteins). Bmf. BH2. Rohon-Beard (RB) cells are an interesting example of this phenomenon. Elevated levels of unhealthy. [60]). Noxa. and Rodriguez and Driever [19]). zebrafish and other vertebrates. e. addition to the GCL and INL. and BH4). the intrinsic apoptosis pathway in mammals [62–64]. and Mcl-1) out vertebrate evolution. The opment in a caspase-dependent manner. Bim. Bad. Bcl-w. 51]. see Abdelilah et al. misplaced. readers are encouraged to consult Pyati et al. Pro-apoptotic family mem- apoptotic ‘‘hot spot’’ has been characterized in the ven. In both birds and mammals an possess all four BH domains.g. although only subfamily serve as apical sensors for various apoptotic apoptosis was observed in the statoacoustic ganglion in stimuli and are capable of promoting apoptosis through agreement with previous reports in birds and mammals activation of the multidomain pro-apoptotic subfamily and/ [57]. [59]. lens-foreign cells that could interfere with subsequent Off-target apoptosis is most pronounced in the CNS and development [48. It has been suggested that apoptosis aids in iments have suggested that many zebrafish genes have separation of the lens from the ectoderm [48]. Two more recent studies have failed to con. vesicle [49]. Bax. 24 hpf as the developing lens is detaching from the surface In addition to forward genetic screens. Bak. Although the onset and Mutations that affect cell death in many other organs and progression of retinal apoptosis in zebrafish is reminiscent tissues have also been described (reviewed in Pyati et al. and BH3 domains. mutants does not stem from defects in the core apoptotic Developmental apoptosis in non-neuronal cell types has machinery. [18]. Furutani-Seiki et al. Bid. [58]. Bcl-xL. knockdown exper- ectoderm. ‘‘BH3-only’’ proteins (e. on pro-apoptotic proteins bind to a hydrophobic groove types that affect developmental apoptosis. The mentation [52]. The cell-intrinsic apoptosis pathway brates such as teleosts and amphibians but are not present in birds or mammals. bers are categorized as multidomain proteins (containing tromedial wall of the developing inner ear adjacent to the BH1. Bcl-2. totic cells in the developing ears of both zebrafish and Puma. It is probable that aberrant cell death in most of these zebrafish is significantly less. In zebrafish. interpretation of these results is greatly firm that apoptosis is a significant factor in separation of complicated by recent findings that morpholino antisense the zebrafish lens from the surface ectoderm [50. developmental apoptosis underlies pro. a process that shares embryos in which developmental cell death is perturbed. phase encompasses cells of outer nuclear layer (ONL) in Fadool et al. family members (e. 123 . RB cells exist only transiently during The zebrafish Bcl-2 family early development and their sensory functions are soon replaced by neurons of the dorsal root ganglia. The first phase is restricted to the ganglion cell layer cell death in the central nervous system (CNS. Hrk. and Bok) and auditory ganglion [56]. 51]. Apoptosis appears Bcl-2 family can be divided into three subfamilies based to be driven in part by loss of the neurotrophic factor ntf3 on apoptotic function and the presence of up to four distinct and may also be regulated by voltage-gated Na? current a-helical motifs known as Bcl-2 homology (BH) domains [53–55]. TUNEL-positive cells in the lens appears to be mediated through tp53 activation. The Bcl-2 gene family serves as the chief regulator of RB cells are eliminated during the first 4 days of devel. [60]. oligonucleotides and other knockdown technologies often Apoptosis may instead be used to clear undifferentiated or induce nonspecific off-target apoptosis in zebrafish [61].

Clarp1 NM_194399 11 123 . 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. Tnf-alpha. tnf. 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. NM_212859 19 Yes [172] TNF-a_v1.Apoptosis (2010) 15:331–349 335 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. 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. 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-a_v3 TNF (TNFSF2) tnfb TNF2. tnf-alpha. casp10-like XM_680338 9 Caspase-9 caspase-9 NM_001007404 23 Yes [24] Caspase-10 LOC795066 caspxb. NM_001024447 15 Yes [24] TNF-a_v2 CD95L/FasL (TNFSF6) faslg FasL NM_001042701 16 Apo2L/TRAIL (TNFSF10) tnfsf10l DL1a.

zfMcl-1a NM_131599 19 Yes [31] Mcl-1 mcl1b NM_194394 16 Yes [31] BCL2L10 bcl2l10 zNR13. zfBcl-xL NM_131807 23 Yes [31] Bcl-w (BCL2L2) – Mcl-1 mcl1a zfMLP1. bax NM_131562 3 Yes (ZFIN) (Pro-Apoptotic) Bax baxb zBax2 NM_001013296 3 Yes [31] Bak – Bok boka zBok1. atr XM_691071 2 231J24.1 123 . mcl1l NM_194398 18 Yes [31] Bfl-1 (BCL2A1) – BCL2L12 – Bcl2 Bax baxa zBax1. birc3 NM_194395 21 Yes (ZFIN) cIAP2 (BIRC3) – XIAP (BIRC4) xiap birc4 NM_194396 14 Survivin (BIRC5) birc5a bir5a. 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. survivin.336 Apoptosis (2010) 15:331–349 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. Nrz. 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. zfBLP1. NR-13.

cip1. 62. one member date suggests that zebrafish may be a better system for of the multidomain pro-apoptotic subfamily (Bak). Zebrafish multidomain Bcl-2 family members show multidomain Bcl-2 proteins have been described (buffy and the highest degree of conservation with their mammalian debcl) and C. 66. and modeling the mammalian intrinsic apoptosis pathway than three members of the BH3-only subfamily (Bcl-G.Apoptosis (2010) 15:331–349 337 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. and simple metazoans such as Drosophila. It is probable that additional pro-apoptotic Bcl-2 proteins in zebrafish and mammals. Bfk. 73]. elegans. 123 . 72. ily. where one multidomain Bcl-2 pro- counterparts. 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. 67]. larly among members of the divergent BH3-only subfam- The only exceptions are three members of the anti-apop. particu- have been described in zebrafish (Table 1) [31. waf1 XM_001923789 22 Yes (ZFIN) p53 tp53 p53. within the genome an important criterion for establishing There is a great deal of functional conservation between orthology [31. The wide range of Bcl-2 family members identified to totic subfamily (Bcl-w. 1) [62. Bfl-1. 66–71]. and BCL2L12). 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 zebrafish Bcl-2 proteins remain to be described. making conserved synteny have been described (Fig. brp53. Members of the BH3-only subfamily display tein (ced-9) and two BH3-only protein (ced-13 and egl-1) greater sequence divergence. where only two Hrk). ptdsr.

66. The diagram illustrates core components of the receptors but this activity has not yet been formally demonstrated for intrinsic and extrinsic apoptosis pathways in evolutionarily diverse faslg. tosis. Mutation of this is either functionally diverged or not a true ortholog [66]. domain blocks the ability of zebrafish bida. pmaip1/noxa. bmf2. [24] although it remains unclear if some instances. interactions may not be established for all members bida is responsible for mediating this interaction of a given family. bcl2l11/bim. Interestingly. leading to speculation that it proteins requires an intact BH3 domain. In ‘‘confirmed’’ based on Eimon et al. Zebrafish bida does not share conserved mammals. bbc3/ proteins. birc5a. confirming a high degree of 123 . and bbc3/puma to induce apoptosis in apoptosis in mammalian cells. elegans. Knockdown of bad also prevents apop- Conversely. 1 Comparison of Apoptotic Pathways in C. anti-apoptotic activity has been demonstrated for species. bmf1. As in transporter [75]. it is capable of sensitizing embryos to induces caspase-3 activation and apoptosis [31. inhibition of bbc3/puma. Similarly.338 Apoptosis (2010) 15:331–349 Fig. baxa. and tnfsf10l3 have Overexpression of zebrafish BH3-only subfamily members embryos [66]. and bnip3l and the multidomain pro-apoptotic sub. bcl2l11/bim. In zebrafish. tnfsf10l. and birc5b but not the other zebrafish IAPs. Zebrafish BH3-only proteins are capable of triggering pmaip1/noxa. the pro-apoptotic function of zebrafish BH3-only synteny with mammalian Bid. Drosophila. bik. all been shown to trigger apoptosis through cell surface death Fish. bad and bida are notably weak inducers of apop- puma. red arrows indicate interactions with strong birc2. For example. or baxb using totic cell death in the CNS of embryos that are defective for antisense morpholino oligonucleotides protects embryos glucose uptake due to inhibition of the slc2a1 membrane from apoptosis in response to c-irradiation [31]. c-irradiation [66]. although bad does not strongly induce family members baxa and baxb in early stage embryos apoptosis on its own. tnfsf10l2. A link supporting evidence based on co-immunoprecipitation studies and/or between the extrinsic and intrinsic pathways in zebrafish is considered antisense morpholino oligonucleotide knockdown experiments. Among the zebrafish pro-apoptotic Bcl-2 bida. and Mammals. 74].

overexpression of bad alone strongly induces and does not bind at all to pmaip1/noxa [66]. zebrafish bim. indicating that the zebrafish immunoprecipitation. Mam. and -7) through pro- Zebrafish anti-apoptotic Bcl-2 subfamily members also teolytic processing. BH3 peptides are capable of interacting with endogenous In mammals. effector caspases (e. Conversely. Mcl-1-/. and Puma by Bak-/. One such factor is cytochrome c. bmf2. which facilitates binding of 88].double knockout MEFs.12 cells embryonic fibroblasts (MEFs). It is assumed that specific patterns of interaction pro-apoptotic Bcl-2 proteins in zebrafish and mammals. two other by ectopic expression of BH3-only subfamily members and mitochondrial factors—Smac/Diablo and the serine prote- by c-irradiation [31. knockdown of bcl2 Although the zebrafish apoptosome has not been char- has no effect on embryo viability [31]. In response to apoptotic stimuli. the site that would ostensibly allow for proteolytic activation apoptosome activates caspase-9. In mammals. Mitochondrial fragmentation is observed and show no embryonic defects [84]. and bbc3/puma. Overexpression of zebrafish bcl2. meabilization and indirectly promote apoptosis by rafish embryos. In addition to cytochrome c. Additional apoptosis in embryos [66]. bad. AKT-dependent phosphorylation of serine 87 [80]. shown to bind directly to human Bim. 79]. While it remains the cytosol. exist between individual Bcl-2 family members. In phorylation. none of these [86]. the BH3-only protein Bid members induce mitochondrial outer-membrane perme- plays a unique role as a bridge between the extrinsic and abilization and trigger release of pro-apoptotic factors into intrinsic apoptosis pathways [82]. and localization to microtubules [81]. but the Mammalian Bad is regulated by phosphorylation of three exact hierarchy of these interactions is controversial [87. which in turn activates the by the extrinsic apoptosis pathway [31]. during early development. COS-7 cells [85] and zebrafish bcl2 promotes survival chrome c release in mitochondria isolated from mouse following growth factor withdrawal in murine FL5. Simultaneous knockdown of both ase Omi/HtrA2—are released following membrane per- mcl1a and mcl1b significantly decreases viability in zeb. In contrast. These loss-of. conserved serine residues. Transfection of COS-1 cells with anti-apoptotic Bcl-2 subfamily members retain sufficient zebrafish bad induces DNA fragmentation and chromatin conservation to function in mammalian cells. protein binds strongly to the BH3 domain peptide from when they are changed to alanine to prevent phosphory. it appears highly homologous to its function phenotypes in zebrafish are similar to those mammalian counterpart. and bbc3/puma induce cyto. pro-apoptotic Bcl-2 family binding site [66]. 66]. This observation indicates that work is required to reconcile these observations and the protein’s pro-apoptotic activity is normally inhibited by determine if individual Bcl-2 family members exhibit serine phosphorylation during early development.mice are viable (Table 1) [71].g caspase-3.Apoptosis (2010) 15:331–349 339 functional conservation. Bax. which binds unknown if bida fulfills a similar role in zebrafish. Once assembled. the zebrafish bcl2 protein was peptides show activity in mitochondria isolated from Bax-/-. anti-apoptotic Bcl-2 proteins associate murine multidomain pro-apoptotic proteins [66]. pmaip1/noxa. acterized in detail. mammals. Significantly. ylation of serine 69 (leading to proteasomal degradation) [78. binds weakly to bida. but appears to lack an obvious dynein light chain. pmaip1/noxa. in zebrafish bad and appear to play a similar regulatory bmf1.0 [83] while Bcl-2-/. demonstrating that anti-apoptotic Bcl-2 inhibiting a family of anti-apoptotic proteins known as proteins play an important role in promoting survival inhibitor of apoptosis proteins (IAPs) [89]. Activated effector caspases cleave show structural and functional conservation with their multiple major cellular substrates and are responsible for mammalian counterparts. When they are changed to aspartate to mimic phos. Several zebrafish in zebrafish embryos undergoing staurosporine-induced 123 . GST pull-down experiments. bcl2 role. amplifying pro-apoptotic signals from both intrinsic and bcl2l/bcl-xL. Two of apoptosis induced by overexpression of the multidomain these residues (serines 136 and 155 in mice) are conserved pro-apoptotic protein baxa and the BH3-only proteins bida. In the latter example. fails to inhibit baxb and only weakly inhibits bik [31]. mcl1a and mcl1b inhibits apoptosis induced extrinsic pathways. the adaptor Apaf-1 and the initiator caspase-9 to form a sequence analysis reveals a putative caspase-8 cleavage complex known as the apoptosome. similar protein-protein interaction patterns in zebrafish and malian Bim is regulated by ERK1/2-dependent phosphor.mice die around components have been identified in the zebrafish genome embryonic day E4. with pro-apoptotic family members through their BH3 Important regulatory sequences are conserved between domains. -6. lation. full-length zebrafish bcl2 tizing embryos to radiation-induced apoptosis. Zebrafish The zebrafish apoptosome and effector caspases bcl2l11/bim shows conservation of the serine 69 and 87 residues. Initial studies in zebrafish show that bcl2 can inhibit 14-3-3 proteins and promotes cell survival [77]. Orthologs of all major apoptosome observed in knockout mice. Zebrafish BH3 domain peptides from bcl2l10 promotes survival following serum withdrawal in bcl2l11/bim. zebrafish bad is no longer capable of sensi. and bbc3/puma. In contrast. Zebrafish condensation [76].

rafish [104]. 95. which interact with TNF receptors between two different effector caspases in mammals [90].340 Apoptosis (2010) 15:331–349 apoptosis. active increased numbers of apoptotic cells. reduced vasculature. pattern during early embryogenesis is similar to that of ponent of the zebrafish apoptotic machinery [91]. disorganized angiogenesis. apoptosis to exert anti-apoptotic functions. The apoptosis following overexpression in cultured cells and tom mutation is located in the zebrafish birc2/ciap1 gene zebrafish embryos [24. There are two zebrafish orthologs of cas. Structural information for down of the closely related birc5b/survivin has a much less caspase-3a has been obtained by X-ray crystallography. In birc5a/survivin [102] Knockdown of either birc5a or birc5b addition to the canonical effector caspases. A forward genetic screen for effector caspase targets are broadly conserved between fish zebrafish vascular defects identified the tomato (tom) and mammals. which produces a hemorrhagic phenotype.5 and exhibit extensive hemorrhaging throughout the C-terminal RING domain and one to three N-terminal body. -6. Zebrafish of caspase-9 in zebrafish [26]. The finding that (TRAF2) and 2) the E3-ubiquitin ligase activity of the zebrafish caspase-3a has low but significant activity RING domain. 97–101]. diffuse vascular endothelial apoptosis. Survivin-/. are important for apoptosis following overexpression [92]. caspases cleave many of the same protein substrates known Several recent publications show that the IAP family in mammals. Zebrafish caspase-9 can rapidly trigger Eight IAPs have been identified in humans and at least four caspase-3 activation and apoptosis when overexpressed of these have orthologs in zebrafish (Table 1) [71. 103]. through TRAF2 and regulate TNFa-mediated activation of Caspase-3 enzyme activity is detected in zebrafish embryos the prosurvival NF-jB pathway through polyubiquitination following overexpression of caspase-3a and treatment by of receptor-interacting protein 1 (RIP1) [2. knock- form of caspase-3 [24. A cycloheximide [21. and increased baculoviral IAP repeat (BIR) domains (hence the alternate pericyte-endothelial gaps [107]. vin. orthologs of the IAP inhibitors Smac/Diablo and Omi/ Multiple effector caspases have been identified in the HtrA2 have also been identified but remain to be func- zebrafish genome (Table 1) [24. 90]. 25]. 31]. family members are dedicated inhibitors of apoptosis [94]. 109]. Endothelial cell apoptosis has been observed tory cell death mediated by caspase-1 in mammals [93]. and more with mammalian caspase-1 have been reported to induce generally endothelial cell apoptosis. -8. This may in fact normal vascular development in mammals as well as zeb- be more akin to pyroposis. Although there are no designation BIRC—BIR-containing proteins). two novel also triggers apoptosis in several other tissues including zebrafish caspases (caspase a and caspase b) containing hematopoietic cells and the neural tube [102. has also been reported to play a role in endothelial cell 3a. Activity assays utilizing The closely related mammalian c-IAP1 and c-IAP2 genes caspase-specific bioluminescent substrates confirm that are represented by a single ancestral birc2/ciap1 gene in both c-irradiation and staurosporine cause strong activation zebrafish and other lower vertebrates [96]. Inhibitors of caspase-8 can rescue the birc2/ against a mammalian caspase-6-specific substrate has led ciap-/. second member of the zebrafish IAP family. a novel form of proinflamma. including PARP and 14 novel human cas. caspase-9. and pro-apoptotic Bcl-2 family members has survival [95. 25]. and -9 substrates [25]. although not all tosis pathway triggers disruption of the mitochondrial outer. this may be due to the high degree of 123 . birc5a/survi- fish following c-irradiation and overexpression of caspase. Surprisingly. 96]. Activation of caspase-3 in zebra. vas- pase-3 and at least one (caspase-3a) is capable of inducing cular regression. mutation. Knockdown of birc5a/survivin results in been confirmed by immunohistochemistry using a cross. 102]. 71]. membrane [22]. N-terminal pyrin domains and sharing greatest homology Several lines of evidence indicate that IAPs. These observations suggest that homeostasis in zebrafish. partitioned mammalian c-IAPs. The anti- shows strong activity toward the mammalian caspase-3 and apoptotic function of birc2/ciap1 in endothelial cells is -7 substrate Ac-DEVD-MCA and weak activity toward dependent upon 1) binding to TNFR-associated factor 2 caspase-1. but selective inactivation of Survivin in endothelial cells IAPs have been described in both invertebrates and using the cre-lox system results in embryos that survive to vertebrates and are characterized by the presence of a E12. and endothelial cell apoptosis [96]. in mouse embryos beginning at E14. plays a critical role in endothelial cell survival and vascular pase-3 substrates [21. and reactive monoclonal antibody against the cleaved. Recombinant caspase-3a and produces a nonfunctional protein product.knockout mice die by E3. in zebrafish embryos [24]. suggesting that activation of the intrinsic apop. Zebrafish effector tionally characterized (Table 1). These observations are consistent with to the suggestion that it plays dual roles. dramatic effect on angiogenesis although its expression providing a unique level of detail for this important com. IAPs can reports of vascular defects in mice deficient for c-IAP1 or interact directly with caspases and other effectors of c-IAP2 [108.5 [106].5 and inhibition of this process by the anti-apoptotic Bcl-2 protein results in abnormally dense and disorganized networks of small Zebrafish IAP proteins and IAP antagonists vessels [105].phenotype.

Bid. caf1b. also seen in tp53M214K (sometimes referred to as tp53e7). [86] have used the zebrafish tp53M214K mutant develop malignant peripheral nerve sheath tumors at to better understand p53-independent apoptotic responses approximately 8. the zebrafish counterpart of the human p53 degree of protection is observed when embryos are pre.5 months of age. the TNFR1 ligand characterized in zebafish. Apaf1). but not in embryos from the tp53M214K mutant ultraviolet irradiation. a brain-enriched microRNA lowing induction and directly interact with both pro. 113] and increase tp53 protein roblastoma cell line SHSY5Y. Puma has allows for functional redundancy in some contexts [99. In stressed cells. and FLJ11339). and embryos rescues the knockdown phenotype and inhibits compromised DNA replication in mutant lines such as apoptosis following DNA damage. Mounting evidence indicates Several recent studies have used zebrafish to identify and that transcription-independent activities also play an validate novel mechanisms of p53 regulation that are important role in p53-induced apoptosis. have not been functionally characterized. a pharmacological inhibitor of p53 following DNA damage and functions to antagonize [119]. Specifically. Le et al. zebrafish tp53 autoregulates its own chek1-knockdown embryos are TUNEL positive. Chen et al. initially identified in a high-throughput Stabilized p53 accumulates in the nucleus and can directly in vitro screen using a human cell line. the tp53M214K line begins to Sidi et al. restore sensitivity to c-irradiation-induced apoptosis in nificant roles in apoptosis in zebrafish [120. The related tran. confirming tp53 is a and gain important insights into the function of caspase-2. Knockdown of checkpoint kinase 1 (chek1) was found to scription factors tp63 and tp73 do not appear to play sig. 123 . isoform D133p53. caspase-3 expression [29] and promotes transcription of a diverse activation is not observed and cell death cannot be blocked array of target genes. using a morpholino zebrafish line with a dominant negative mutation in the targeting the D113p53 5’-UTR.Apoptosis (2010) 15:331–349 341 conservation between these two family members. critical tumor suppressor protein in fish. including cdkn1a/p21 [29]. NR2F2. which can be rescued by simultaneous knockdown of tp53 [29]. inducing tp53 expression in zebrafish embryos [126]. Overexpression response to DNA damage in zebrafish. and pinball eye [116–118]. p53 broadly conserved throughout vertebrate evolution. apoptosis in primary human lung fibroblasts and the neu- fish tp53 transcription [29. a Knockdown of endogenous D113p53. iated by stimulation of the intrinsic apoptosis pathway. Resistance to DNA damage-induced apoptosis is apoptosis. Other ubiquitin ligases known to The tumor suppressor protein p53 is a multifunctional negatively regulate p53 in mammals—such as COP1 [124] transcription factor that plays a central role in determining and Pirh2 [125]—are present in the zebrafish genome but how cells respond to stresses such as DNA damage. Seven novel acti- the latter case. Knockdown of tp53 blocks apoptosis arising miR-125b leads to increased apoptosis in wild-type from a wide range of stressors.g. tp53M214K embryos. addition to reduced apoptosis. increases the sensitivity of tp53 locus that destroys transcriptional activity [113]. in part through upregulation of bcl2l/bcl-xL. can translocate to the mitochondrial outer membrane fol. p53-dependent apoptosis is primarily med. Osr1. A similar that D113p53. This hypothesis is supported by the finding that tosis in mammals based on data from knockout mice [123] simultaneous depletion of c-IAP1 and c-IAP2 in human and bbc3/puma appears to play a similarly essential role in umbilical cord vascular endothelial cells (HUVECs) sen. [127] identified miR-125b. and bbc3/puma [31]. searching for miRNA response elements that have been The tp53 gene is a critical mediator of apoptosis in conserved between zebrafish and humans. ligase mdm2 serves as a primary negative regulator of tp53 activation. Activation of p53 can lead to a variety of outcomes. Injection of synthetic miR-125b duplex into zebrafish bition of the DNA crosslink repair gene fancd2 [115]. which baxa [113]. TFAP4. by anti-apoptotic members of the Bcl-2 family [111. In dependent apoptosis in zebrafish [116]. as a potential negative regulator of p53. 121]. Knockdown of zebrafish mdm2 results in Zebrafish p53 and the intrinsic apoptosis pathway extensive apoptosis. [128] show flathead.and (miRNA). zebrafish [31]. embryos. Hes1. camptothecin treatment [29]. 112]. SFRS10. in piy mutant embryos shows that two genes known to p53 is stabilized and converted from a latent to an active mediate the DNA damage response upstream of p53 in form. been identified as a key mediator of p53-dependent apop- 100]. mdm2 and by bcl2l/bcl-xL overexpression or bbc3/puma knockdown. perp [122]. although dying cells in As in mammals. A variety of DNA of miR-125b reduces p53 levels and inhibits p53-dependent damaging reagents have been shown to upregulate zebra. is activated in a p53-dependent manner treated with Pifithrin-a. Surprisingly. vators of p53 (Hey1. the E3 ubiquitin [96]. Puma. As in mammals. line. including c-irradiation [32]. mammals—ataxia telangiectasia mutated (atm) and including cell cycle arrest (allowing for DNA repair) and checkpoint kinase 2 (chek2)—are also essential for tp53- apoptosis (to eliminate cells damaged beyond repair). knockdown of levels [114]. are capable of regulate the expression of many pro-apoptotic genes (e. Noxa. Upstream regulation of tp53 has also been sitizes these cells to killing by TNFa. In zebrafish. In embryos to c-irradiation-induced apoptosis. Epistasis analysis hypoxia. and oncogenic signaling [110]. inhi.

2). Orthologs of both only a single ortholog of the closely related death receptors RAIDD (cradd) and PIDD (LOC571033) have been designated DR4 and DR5 in humans and other primates identified in the zebrafish genome (Table 1) and overex. The diagram illus. The roles of the RAIDD and receptor ligands in zebrafish and other teleosts. (appa) have been identified based on phylogenetic analysis Fig. An although it does not appear to be required for cell death in extracellular fragment of b-amyloid precursor protein most contexts [130]. [134]. tnfsf10l3. however. (APP) has recently been shown to act as a ligand for DR6 vation has not been fully elucidated. and tnfsf10l4). 2 Zebrafish Death Receptors and Ligands. and the initiator caspase-2 are critical for radiation-induced apoptosis in tp53M214K/chek1-knock. and APP pathway is unclear. co-immunoprecipitation studies and/or antisense morpholino oligo- trates all TNF and TNFR superfamily members that have been nucleotide knockdown experiments. pression of casp2 on its own is sufficient to induce Recent studies have shed considerable light on death apoptosis in embryos [24]. tnfsf10l2. that respond to DNA damage by activating a number of Death receptors are characterized by the presence of a downstream proteins including p53 [129]. is a matter of long. ATR. CD95/FasL (faslg).342 Apoptosis (2010) 15:331–349 Epistasis analysis demonstrates that zebrafish orthologs The cell-extrinsic apoptosis pathway of ATM. Gray arrows indicate interac- reported to date in zebrafish. Apo2L/TRAIL their requirement in the novel Chek1-suppressed caspase-2 (tnfsf10l. Red arrows indicate ligand. Experiments in HeLa cells suggest that a similar Chek1-suppressed caspase-2 pathway exists in The extrinsic apoptosis pathway is engaged when certain mammalian cells defective for p53 activity. This arrangement of receptors and ligands is well that incorporates the adaptor proteins RAIDD and PIDD conserved in mammals. In mammalian systems caspase-2 has DISC [132]. Orthologs PIDD orthologs in zebrafish have yet to be studied and of TNF (tnfa and tnfb). Official zebrafish gene designations are tions that are hypothesized to occur based on studies in other given in lower case italic type and corresponding mammalian vertebrate species but remain to be experimentally verified in homologs are given in upper case bold. cytoplasmic tail that plays a central role in formation of the standing debate [3]. The precise role conserved *80-amino-acid death domain motif in the of caspase-2 in apoptosis. although it is thought and drive axonal degeneration and neuronal death in mice to involve a protein complex similar to the apoptosome [133]. although many species possess (designated the PIDDosome) [131]. The human genome contains six death been linked to both the intrinsic and extrinsic pathways. zebrafish. receptors and five cognate TNF ligands (Fig. Zebrafish death receptors and ligands down embryos. ligands of the TNF superfamily bind to specialized mem- ATM and ATR are well-characterized apical kinases bers of the TNFR superfamily known as death receptors. The mechanism of caspase-2 acti. The zebrafish homolog of APP is included as a potential receptor interactions that have been confirmed in zebrafish by ligand for tnfrsf21/dr6 based on recent evidence in mammals 123 .

The finding that both hdr tions [146]. but not tradd. tinctive QACQG active-site motif but lacks N-terminal ment of the adaptor protein FADD leads to DISC recruit. [137] describe a [145]. two death domain-containing taining N-terminal death effector domains (DEDs) and a receptors designated hdr [141. and -1 in receptor without a zebrafish counterpart. an enzy- that tnfa binds selectively to tnfrsf1a. although the mammals—blocks Apo2L/TRAIL-induced apoptosis in identification of a zebrafish ligand with homology to TL1A zebrafish embryos [24]. In of TNFR1. Casp8l1 contains the dis- through one of two distinct signaling complexes. and CD30L in an unrooted phylogenetic tree receptor signaling pathways through distal signaling com- [135]. zebrafish possess a clear ortholog of c-FLIP caspase-8 is positively controlled by Cullin-3-mediated (cflar) that contains N-terminal DEDs and an enzymati- polyubiquitination and p62-dependent aggregation [143] cally inactive caspase cataltic domain. con- (Table 1) [24]. has been described [24. the intronic structure of bind TRADD and mediate immune-stimulatory activity. other Apo2L/TRAIL and RANKL sequences [135]. In mammals. [135] suggests such an ortholog may yet be found. (tnfrsf1a. lta is distinct from human LTa and it clusters with OX40L. DR3 remains the only mammalian death A (CrmA)—which inhibits caspase-8. DR5. the zebrafish ortholog matically inactive relative of caspase-8 and -10 [144]. Activated death receptors mediate downstream activity LOC795066. TNFR1 and DR3 to the zebrafish tnf locus. domains. Co-immunoprecipitation studies confirm and cellular FLICE-inhibitory protein (c-FLIP). Additional caspase-8 related genes are present in zeb- rafish (Table 1) [24. Components of the zebrafish DISC and three genes containing an N-terminal caspase recruit- ment domain (CARD) in place of the DED motifs (caspxa. Zebrafish casp8a can mediate apoptosis fol- and tnfrsfa bind Apo2L/TRAIL orthologs and are essential lowing death receptor activation in mouse embryonic for Apo2L/TRAIL-induced apoptosis in zebrafish argues fibroblast (MEF) cells that lack endogenous caspase-8 strongly for categorizing them as homologs of DR4 and [146]. fas. and zgc:194469). (as well as caspase-10 in human cells) [132]. tnfsf10l3 are more structurally related to Apo2L-like—a Reverse genetic analysis has been used to interrogate the second copy of Apo2L/TRAIL found in avian species but requirement for fadd and tradd in the zebrafish DISC. In addition. casp8l2. Of the four zebrafish Apo2L/TRAIL homologs. Clear orthologs of TNFR1 (tnfrsf1a). indicating that the death domain motif CD95/Fas (fas). They contain extracellular domains conserved with its mammalian counterpart. These include casp8l1. Both tnfsf10l and adaptor proteins FADD and TRADD (Table 1) [24. as expected [24]. is sufficient to block extracellular domains [140]. only DR4.Apoptosis (2010) 15:331–349 343 [24. phylogenetic analysis of their death within the DISC [24]. and DR6 (tnfrsf21) have been identified has been sufficiently well conserved between teleosts and based on the arrangement of their cysteine-rich extracel. Engage. and CD95/Fas— novel TNF gene (TNF-N. casp8l2 possesses DEDs but contains a ment and drives proapoptotic signaling through caspase-8 QACRG active site that is more reminiscent of caspase-2. subsequently designated lta) that which signal primarily through FADD—are direct activa- appears similar to mammalian LTa based on its proximity tors of the extrinsic apoptosis pathway. A probable zebrafish TL1A contrast. and p38 MAPK pathways other teleosts remains unclear. and tnfrsfa) and fadd in human cells rafish and mammals. plexes that are nucleated by FADD and TRADD [2]. However. Zebrafish tnfsf10l4 shows the apoptosis induced by overexpression of the Apo2L/TRAIL greatest sequence divergence and only groups weakly with orthologs tnfsf10l and tnfsf10l3 in zebrafish embryos [24]. FADD directly that are most similar to CD95/Fas and death domains that associates with casp8a through homophilic DED interac- are most similar to DR4 and DR5. In contrast. Cross talk is possible between these two distinct death GITRL. 135–139]. 142] and tnfrsfa (previously QACQG active-site motif characteristic of mammalian known as Ovarian TNF Receptor or otr) [139] have been caspase-8 and -10. both proinflammatory cytokines and antiapoptotic factors teine residues [135]. 71]. triggers apoptosis. and conserved synteny with mammalian genes A single zebrafish ortholog of caspase-8 (casp8a). Savan et al. Several described in zebrafish. JNK. Transient overexpression of zebrafish death receptors Death receptors are also well conserved between zeb. including the pivotal mammalian Apo2L/TRAIL [24]. not mammals—due to additional cysteine residues in their Inhibition of fadd. Activation of In addition.’ which induces transcription of phylogenetic analysis and the presence of conserved cys. DEDs. The cowpox virus serpin cytokine response modifier DR5 (Fig. These receptors have less clear-cut lines of evidence show that zebrafish casp8a is functionally mammalian orthologs. -10. The status of LTa in zebrafish and through the IKK/NF-jB. Zebrafish orthologs have been identified for most com- tnfsf10l2 exhibits the greatest structural similarity with ponents of the DISC and Complex I. 2) [24]. 146]. engagement of the adaptor protein TRADD leads ortholog (LOC560966) has also been described based on to assembly of ‘Complex I. 146]. mammals to enable cross-species protein interactions lular domains. hdr. Overexpression of and negatively controlled by the deubiquitinase A20 [143] cflar in zebrafish embryos prevents induction of apoptosis 123 .

Bcl-2 proteins regulate the release of pro-apoptotic mole- iod. T and B cells do not to be important in mammals. models cannot replicate many aspects of apoptosis known lation [3. In spite of their advantages. 155]. role in Drosophila apoptosis but are regulated primarily via and tnfsf10l3) are expressed in regions of the developing increased expression rather than mitochondrial sequestra- CNS. elegans and consists reported similar findings in zebrafish embryos following of only a single member lacking a death domain (eiger) in morpholino knockdown of hdr. and Hid (RGH) proteins— zebrafish [152]. elegans and Drosophila. [141] found that hdr is debcl) [159]. Two studies demonstrate that the zebrafish death identified in humans [62] versus three in C. while in mammals receptor signaling may play a role in numerous zebrafish they serve as the chief regulators of the intrinsic pathway. making analysis of the normal part of embryogenesis and adult tissue homeostasis extrinsic pathway in these cell types by morpholino is often specific to organs and tissues that lack analogs in knockdown techniques impractical.and B-cell homeostasis. cytochrome c lateral line. 164]. [151] invertebrate species. selectively induces apoptosis in invertebrates and mammals. 157]. there are fundamental differ- observations. elegans cells similar to those of the mammalian inner ear and are and its role in Drosophila appears to be extremely limited known to undergo constant. Unlike mammals. Fundamental differences are also apparent mammals the expression of death receptors and ligands in the extrinsic pathway. These mechanosensory organs contain hair does not play a role in apoptosome formation in C. An even more extreme case is the TNFR specifically expressed in the hematopoietic lineage and superfamily. apoptosis-mediated turnover in [159. Tnfrsfa and the Apo2L/TRAIL homologs cules such as cytochrome c. apoptotic pathways have evolved to an integral role in zebrafish T. it is interesting to note that overexpression of ences in the way apoptotic pathways function between tnfsf10l3. tnfrsfa is also expressed in the notochord during this per. tissues [24]. 165]. Drosophila Bcl-2 proteins the erythroid lineage [24]. The ease with which these mutations in CD95/Fas. 147]. [142] superfamily is not present at all in C. This an accumulation of red blood cells. cal conditions. Long et al. functional counterparts of Smac/Diablo—play a central tnfrsfa. The closely related death receptor mammals versus C. and caspase-10 are systems can be genetically manipulated has been instru- responsible for autoimmune lymphoproliferative syn. and tnfrsf21/dr6) and ligands (tnfsf10l. molecules are regulated under both normal and pathologi- Knockout studies in mice confirm that the extrinsic path. Additionally. apoptosis arises from have used a stable transgenic line expressing bcl2 under the disease pathologies that cannot be modeled in nematodes control of the rag2 promoter to show that apoptosis plays and flies. Eiger triggers cell death through a 123 . CD95L/FasL. The Reaper. Biological roles of the extrinsic apoptosis pathway in zebrafish Concluding remarks Although the extrinsic pathway is critical for normal Invertebrate model organisms such as C. Their role in zebrafish remains unknown. receptor hdr plays a homeostatic role in at least one non. and ced-13) [158] and two in Drosophila (buffy and lymphoid cell type. In the context of these Drosophila [161]. while mutations in caspase-8 cause deficiencies in of the apoptotic machinery and understanding how these both lymphocyte proliferation and activation [2. appear to be peripheral players responsible for fine-tuning Embryonic gene expression patterns suggest that death stress-induced apoptosis [162. invertebrate way has widely conserved roles in immune system regu. In humans. but in tion [162. elegans (egl-1. ced-9. as is fadd. study apoptosis [154. (particularly CD95/Fas and DR6) in the CNS is thought consistent with its roles as an inhibitor of death receptor. Grim. In zebrafish. [133. and Omi/HtrA2 tnfsf10l2 and tnfsf10l3 are detected in neuromasts of the from the mitochondria. Finally. more than 25 Bcl-2 superfamily members have been types. elegans and functioning of the mammalian immune system. but a far greater degree of complexity and diversity in mam- additional transgenic or mutant lines will be required to mals and other vertebrates (Fig. For exam- verify a specific role for the extrinsic pathway in these cell ple. 148. 163]. 153]. In other cases. In addition to the hematopoietic lineage. hdr is This difference may be linked to the increased importance strongly expressed in the developing notochord during of mitochondria as activators of the intrinsic pathway in early development. Kwan et al. the hdr ligand. 149]. 1) [156. mental in identifying and characterizing core components drome. Smac/Diablo. tnfsf10l2. several death receptors (tnfrsf1a. comprising approximately 30 members in used transgenic zebrafish expressing a dominant negative humans including six death domain-containing receptors hdr version to demonstrate that receptor inhibition leads to responsible for engaging the extrinsic pathway [160]. In mammals. it remains Drosophila have been used widely and successfully to unclear if it plays a similar role in zebrafish. Finally.344 Apoptosis (2010) 15:331–349 by the Apo2L/TRAIL orthologs tnfsf10l and tnfsf10l3 [24]. Langenau et al. Apoptosis that occurs as a develop until 3 dpf or later [150]. to influence a variety of neuropathological processes mediated apoptosis in mammals.

Wienholds E. as demon. that future apoptosis research in zebrafish will continue to scription of antimicrobial peptide genes following bacterial exploit the strengths of this model system. 10. In addition to preserving the reverse genetic techniques. raising the possibility that zebrafish produced by retroviral infection of in vitro-cultured sperm. a vertebrate model of apoptosis been reported only for a handful of the apoptosis-related that is genetically tractable. and exposure to toxins. Bhat GK (2008) Caspases . the zebra. Plasterk RH ring not only during embryonic development but also in (2002) Target-selected inactivation of the zebrafish rag1 gene. 1. 127. Schulte-Merker S. as this review apoptosis pathways. Dower N. zebrafish: zinc-finger nucleases. as these 5. The failure of large-scale forward shows. Walderich B. The value of such genetic tools is nicely than invertebrates on a number of levels. Li L. Dixit V. Solnica-Krezel L. 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