(0–45 minutes postfertilization). When injected at this stage of development, the material is distributed equally after each cell division into every cell of the developing animal. For in vivo gain-of-function experiments, mRNA is transcribed in vitro from a zebrafish expression vector, capped, and polyadenylated. Injection of this mRNA into one-cell stage embryos results in high levels of the gene product in every cell of the animal, and the effects can be monitored for approximately the first 24 hours of life. Conversely, transient loss-of-function experiments can be performed by injecting modified antisense oligonucleotides called morpholinos. These 25-mer oligos, which are commercially available, are capable of blocking either the translation or splicing of an mRNA, depending on the targeted site. Because of their chemical modification, morpholinos are very stable and can remain in the embryo for up to 3 to 5 days, continually knocking down levels of the targeted gene product.

2.2.2. In situ hybridization and immunohistochemistry

Two molecular techniques that can be powerfully used in zebrafish for studying localized gene expression and localized protein expression are in situ hybridization and immunohistochemistry, respectively. For at least the first

5 days of development, in situ hybridization using in vitro transcribed antisense riboprobes can be performed in whole animals to discover patterns of gene expression within developing tissues and to analyze the effects of gainor loss-of-function experiments. Additionally over the same time period, immunohistochemistry can be performed to examine subcellular distribution or tissue localization of specific proteins. Immunohistochemistry and Western blotting are also accepted as the gold standards for assessing levels of protein knockdown in loss- of-function experiments.

2.3. Forward genetic screening

A major strength of the zebrafish over other vertebrate model systems, including the mouse, is that unbiased forward genetic screening can be performed in sensitized genetic backgrounds, even in moderately sized laboratories. The mutagen of choice used in the largescale developmental screens of the mid-1990s is ethylnitrosourea (ENU). This alkylating agent causes random point mutations in the genomic DNA of treated animals, and incubating fertile male zebrafish in ENU causes mutagenesis of their developing spermatogonia. Crossing these mutagenized P0 males to wild-type females

and raising the clutches to adulthood yields F1 animals with individual sets of mutations, many of which will be gene-altering (as shown in Figure 36-2). Subsequently, F1 animals are outcrossed to wild-type fish, and for a standard F3 recessive screen, the F2 families are incrossed to yield embryos that harbor recessive mutations. Investigators can then analyze F3 clutches for their phenotype(s) of interest, knowing that the Mendelian percentage of 25 would be reflective of a true recessive mutation. After identifying a mutant based on an interesting phenotype, positional cloning techniques based on linkage analysis are used to map the mutation of interest and to clone the affected gene.

2.4. Drug and small-molecule screening

The small size, transparency, and permeability of the zebrafish embryo all combine to make this a perfect system for drug and small-molecule screening. Zebrafish embryos take up oxygen through diffusion, and by simply dissolving drugs or small molecules in the water of

a plate holding the fish, an investigator can introduce compounds into the animal’s body. Zebrafish screens have already been successfully performed to discover a novel cell-cycle regulating compound, an angiogenesis regulator, a bone morphogenetic protein (Bmp) signaling inhibitor, and a novel compound that regulates hematopoietic stem-cell number. Because many embryos can be screened at one time in a multi-well format, the high-throughput nature of drug and smallmolecule screening in zebrafish is comparable to that of cells growing in tissue culture.

2.5. Transgenesis

The ability to make transgenic animals is a major strength of the zebrafish system. Recently developed technologies using transposable elements as well as an Isce-I meganuclease strategy have greatly increased the efficiency of germline transgenesis to nearly 50%. By introducing exogenous genes into zebrafish embryos, researchers have already developed cancer models

in zebrafish for T-cell acute lymphoblastic leukemia, melanoma, pancreatic cancer, acute myeloid leukemia, and rhabdomyosarcoma. Other models such as one for neuroblastoma are being actively developed, and each of these models may be an excellent system for tumorspecific cell death–based drug discovery.

2.6. Targeted knockouts

A very recent technology that was developed in Drosophila and applied to zebrafish allows researchers to make targeted knockouts of their genes of interest. This strategy uses hybrid zinc-finger nucleases, which are modified enzymes that cleave DNA at specific 9–base-pair sequences. By injecting mRNA encoding two sets of three zinc-fingers coupled to heterodimeric cleavage domain variants of the endonuclease FokI, researchers can target a specific sequence within their gene of interest to excise DNA. The resulting nonhomologous end-joining repair process frequently introduces microdeletions or frameshift mutations, resulting in early stop codons or missense mutations in the gene sequence. Furthermore, these loss-of- function lesions are highly penetrant and heritable, making it possible to create a stable knockout line for loss-of-function studies in both embryos and adults. Researchers can also create an allelic series for their gene of interest to study the varying effects of different types of mutations on gene function. This new technology should augment the utility of the zebrafish for reverse genetic approaches to inactivate genes and study the ensuing phenotypes. However, it should be noted that there is not yet a conditional knockout system in zebrafish as there is in mice, making the latter still advantageous for use in reverse genetic studies.

3. THE ZEBRAFISH MODEL ORGANISM IN CELL

DEATH RESEARCH

The first systematic observations of altered cell death in zebrafish embryos came from large-scale mutagenesis screens of the mid-1990s in Tubingen, Germany, and Boston, MA. In these screens, scores of mutants with degenerating nervous system cells were isolated based on bright-field opacity of brain and spinal cord tissue, coupled with acridine orange staining to mark dead or dying cells. Since these early studies, researchers in the field have identified and functionally characterized factors important for intrinsic and extrinsic apoptosis, highlighting the large degree of conservation between zebrafish and mammalian apoptotic mechanisms. Additionally, a recent study in zebrafish revealed a new p53-independent apoptotic pathway that can induce

cell death after DNA damage in both zebrafish and human cells. Furthermore, although anoikis has not been formally studied in zebrafish, at least three studies have detailed either endothelial or keratinocyte apoptosis reminiscent of cells undergoing anoikis-induced cell death. Recent work has also uncovered autophagy in developing thymocytes after transgenic over-expression of both Myc and Bcl-2, the first example of this type of cell death mechanism in zebrafish. Necrotic models are infrequent in the zebrafish literature, but two studies indicate that apoptosis is not the sole mechanism of cellular destruction in the embryonic or adult fish in response to defects in pigment biogenesis or pathogen infection. This section highlights recent studies that demonstrate the relevance of the zebrafish system for discovering novel cell death regulators that are conserved in higher vertebrates.

3.1. Intrinsic apoptosis

Three studies have delineated and characterized the conservation of intrinsic apoptotic pathways in zebrafish. The first was a detailed genomic comparison performed by Inohara and Nunez in which they identified the majority of zebrafish apoptotic regulators by species comparison using genomic databases. In a second study, Kratz et al. (2006) used genomic comparisons and splice-site predictions to clone almost all of the zebrafish orthologs of mammalian apoptosis regulators (Table 36-1). They showed that zebrafish intrinsic apoptosis regulators have functions similar to those in mammals, with the p53-Puma axis dictating death after irradiation-induced DNA damage. Additionally, they showed that the zebrafish genome contains bax1 and bax2, but they could not find a true bak ortholog. Through functional studies using mRNA over-expression, they showed that Bax2 functions like human Bak, because its apoptotic potential could only be blocked by Bcl-XL, Mcl1a, or Mcl1b, but not by Bcl-2. Meanwhile, all of the small BH3-only proapoptotic molecules functioned similar to those of their mammalian counterparts, confirming the conservation of intrinsic apoptotic machinery. Finally, in a recent article by Jette et al. (2008), biochemical data elegantly showed that zebrafish apoptotic regulators could interact with mammalian mitochondrial apoptotic proteins to potently induce mitochondrial outer membrane permeabilization (MOMP) with generally similar kinetics to those of their mammalian counterparts. Additionally, this group cloned a true zebrafish bim ortholog, further highlighting the conservation of a wide range of BH3-only molecules in zebrafish. Consistent with

their in vivo effects, Bim and Puma acted most strongly to induce MOMP in the isolated mitochondria system used to assay the apoptotic potential of each molecule. Meanwhile, Bad seemed to act only as a sensitizer to irradiation-induced death, once again in agreement with the mammalian system. Interestingly, zebrafish BH3-only factors have a high degree of similarity to their mammalian counterparts solely in the pro-death BH3 domain, indicating that the other functions of these molecules may have diverged.

3.2. Extrinsic apoptosis

Two different studies have functionally described elements of the extrinsic apoptosis pathway in zebrafish. First, work from Kwan et al. (2006) showed that the zebrafish death receptor gene is a negative regulator of primitive hematopoiesis. Knockdown of the death receptor protein with morpholinos caused an increase in red blood cell number, indicating that extrinsic apoptotic pathways normally regulate cell numbers the erythrocyte population. A second study by Eimon and colleagues (2006) described cloning of the major components from the Fas ligand pathway, the Apo2L/Trail pathway, and the tumor necrosis factor (TNF) pathway. They found that the zebrafish genome contains one fas ligand and four apo2L/trail relatives, which they named death ligands (dl) 1a, 1b, 2, and 3. Additionally, zebrafish contain two different tnf ligand genes, which they named ztnf1 and ztnf2. Genes encoding eight death domain (dd)–containing receptors were identified based on sequence homology and synteny to human orthologs, including a tnr receptor (tnfr), a fas receptor (fas), three nerve growth factor receptors (ngfrs), a hematopoietic death receptor (hdr; described in the Kwan et al. study), an ovarian tnf receptor (otr), and death receptor 6 (dr6). Finally, genes encoding members of the death-inducing signaling complex associating with the activated receptors were identified. These included homolog of the fasassociated death domain (fadd), trail-associated death domain (tradd), and a number of initiator and effector caspases, including caspase-8 and caspase-10.

To functionally characterize the activity of extrinsic apoptosis components, Eimon and colleagues (2006) over-expressed mRNA of the genes hdr, otr, fas, dr6, or tnfr1 and found that injection of the first three mRNAs caused robust apoptosis by 8 hpf, as assayed by immunohistochemistry against activated caspase-3. By the same assay, other mRNAs that induced robust apoptosis between 8 hpf and 24 hpf included fadd, tradd, caspase-2, caspase-3a, caspase-8a, and caspase- 9. Interestingly, over-expression of mRNA encoding the three death ligands (DL1a, 1b, and 2) caused apoptosis

Table 36-1. Analysis of cell death in zebrafish

Intrinsic Apoptosis Pathway Components

Note: List of cloned zebrafish intrinsic and extrinsic apoptotic factors, with human homologs listed on the right.

Adapted primarily from Kratz et al. (2006) and Eimon et al. (2006), Cell Death and Di erentiation 2006, Nature Publishing Group.

specifically in cells of the notochord, the backbone of the zebrafish, at 24 hpf, as assayed by both caspase- 3 activation and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining. DL1b over-expression also caused death of erythrocytes, resulting in lower levels of O-dianisidine staining. These experiments highlight the power of the zebrafish embryo for assessing tissue-specific differences in apoptotic