used these criteria to show that zebrafish with mutations in the transient receptor potential melastatin 7 (trpm7) locus display necrosis in pigment-forming cells called melanophores. This necrosis is dependent on melanin synthesis, as inhibition of the melanin synthesis pathway blocked cell death in mutant embryos. Interestingly, trpm7 is expressed in metastatic melanoma lines, indicating that the role of this gene as a prosurvival factor in melanin-producing cells may potentiate metastasis. Further studies of necrosis have been performed in models of tuberculosis infection, in which disease progression includes the formation of necrotic granulomas. Swaim and colleagues showed that infected cells display cellular breakdown characteristic of necrosis, although this group did not perform molecular analyses to definitively rule out any role of apoptotic mechanisms in cellular elimination.

4. DEVELOPMENTAL CELL DEATH IN ZEBRAFISH EMBRYOS

The majority of the cell death studies described in the preceding section pertain to gainor loss-of-function experiments that examined the effects of changing the balance of cell death factors versus survival factors in zebrafish embryos or adult fish. However, it is also clear that developmental cell death occurs in zebrafish in a regulated fashion, ultimately creating the proper number of cells in each tissue of the body (Figure 36-5). In a study published in 2001, Cole and Ross used TUNEL staining over a series of developmental stages to create a map of cells undergoing apoptosis in the developing zebrafish embryo. From this and previous work, it was established that developmental cell death in zebrafish occurs primarily in nervous system tissues, including the transient Rohon-Beard sensory neurons, all of which die by caspase-9–dependent intrinsic programmed cell death between 18 hpf and 72 hpf. The death of this early sensory neuron population is mediated by a combination of neurotrophin signaling and electrical activity. Intriguingly, elegant studies have shown that the sensory arbors from these neurons persist long after the main cell bodies have died, providing a unique insight into the delayed mechanisms of the complete clearance of dead cells in the nervous system. Other tissues undergoing developmental apoptosis over a series of developmental stages include the eye, the nose (olfactory placode), the brain, the urogenital system, the germ cells, and the tail.

One of the major strengths of studying the zebrafish embryo is the ability to image cell biology in real-time using fluorescent reporter proteins or vital dyes. In a recent study from Peri and Nusslein-Volhard (2008),

Regions of developmental cell death

Figure 36-5. Cell death zones in developing zebrafish embryos. Regions of developmental cell death summarized in a 24-hpf embryo (anterior to the left). Note the diverse range of tissues that die during embryogenesis. All embryos are shown with anterior to the left. Reprinted with permission of Elsevier. See Color Plate 46.

the imaging of neuronal cell death and engulfment by microglia in the brain was achieved with unprecedented clarity. To prevent the accumulation of damaging cell-death products in surrounding cells, microglia (the phagocytes of the brain) quickly digest dying neurons by phagocytosis. Using a double-transgenic zebrafish line with membrane Egfp-labeled microglia and neuronlabeled DsRed, the researchers filmed the process of cellular death in the brain followed by the recognition, engulfment, and phagocytosis of apoptotic cells by neighboring microglia (Figure 36-6). They observed that phagocytic cups in the extended processes of microglia engulf dying neurons and transport them to the microglial cell body to eliminate them. Further work showed that the microglial protein Atp6v0a1 is critical for the digestion of dead neuronal cells within microglia through the formation of phagolysosomes. This exciting study further confirms that the imaging of cell death in zebrafish is a unique and powerful approach to screen for morpholinos, drugs, or mutations that affect fundamental apoptotic processes, such as cellular recognition and digestion after apoptosis.

5. THE P53 PATHWAY

Any discussion of cell death studies would be incomplete without mentioning the p53 pathway and its importance for apoptotic processes during development and in cancer. p53 is activated after cellular stress or DNA damage, and one of its major roles is as a

proapoptotic transcription factor. In zebrafish, a reverse genetics approach called TILLING (targeting-induced local lesions in genomes) was used to isolate a zebrafish p53 mutant that is highly relevant to human cancer. As with most human p53 mutations in cancer, the zebrafish mutant has a single missense mutation in the DNAbinding domain of p53 that renders it incapable of activating transcription. Berghmans and colleagues (2005) showed that this mutant zebrafish line develops malignant peripheral nerve sheath tumors in nearly 30% of animals by 16.5 months of age. Early embryonic assays showed that the animals have a complete loss of DNA damage-induced apoptosis compared with controls. These studies laid the groundwork for the discovery of the Chk1-suppressed apoptotic pathway described earlier, and additional screens using unbiased mutagenesis approaches should further aid our understanding of pathways that can bypass the p53 axis and promote cell death after DNA damage and other stresses.

In addition to DNA damage–based p53 activation, recent work from Robu and colleagues described offtarget activation of the p53 pathway after the injection of

morpholinos designed to knock down gene function in zebrafish. A common toxic effect after injection of some morpholinos is extensive cell death in the brain and nervous system of injected embryos. This unintended side effect has confounded the analysis of apoptotic mechanisms, because investigators had been unable to disentangle the effects of gene knockdown from the effects of morpholino toxicity. However, work from this group showed that co-injection of a p53 morpholino along with a morpholino targeted against the gene of interest can block the unintended nonspecific morpholinoinduced cell death in the brain and nervous system. Thus, researchers can distinguish which tissue patterning and/or cell death mechanisms are likely to be a consequence of morpholino toxicity, and focus on the effects of specific knockdown of the protein of interest. Using a series of morpholinos that had been previously described to cause nervous system cell death, Robu and colleagues showed that simultaneously knocking down p53 rescued the cell death defects without changing tissue-patterning defects that resulted from specific protein knockdown. This work created a new paradigm for

morpholino loss-of-function studies, and it also hinted at a new mechanism for p53 activation by injection of foreign nucleic acids. Though the molecular mechanism(s) for this activation are still unclear, the work suggests that cells have an internal p53-dependent sensor for detecting the presence of morpholino oligonucleotide stress and eliminating cells as a result. Of course, if researchers are studying an antiapoptotic molecule whose specific knockdown actually does trigger p53 activation, other types of controls such as phenotypic rescue or development of a stable genetic mutant become critical for establishing the validity of the observed effects.

6. PERSPECTIVES AND FUTURE DIRECTIONS

In this chapter, we have reviewed studies demonstrating that the zebrafish is a very useful model system for studying cell death processes that are relevant to human development and disease. Although the zebrafish model has been primarily used for studies of vertebrate developmental processes, this system is increasingly being used for studies of disease mechanisms. Technologies such as those employed in the articles we have highlighted are allowing researchers to quickly and effectively attack pressing questions in the field of cell death, and future approaches should further enhance the usefulness of this model system for rapidly assessing drug efficacy and the essential functions of cell death regulators. Zebrafish thus combine the strengths of flies and worms in genetic analysis based on phenotype assessment with the strengths of the mammalian mouse model in its relevance to human disease processes. From a muddy freshwater river in India, the zebrafish has begun to show its stripes as a premier animal model for studying vertebrate cell death pathways.

SUGGESTED READING

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Cole LK, Ross LS. Apoptosis in the developing zebrafish embryo.

Dev Biol. 2001 Dec 1;240(1):123–42.

Eimon PM, Kratz E, Varfolomeev E, Hymowitz SG, Stern H, Zha J, Ashkenazi A. Delineation of the cell-extrinsic apoptosis pathway in the zebrafish. Cell Death Differ. 2006 Oct;13(10):1619–30.

Feng H, Stachura DL, White RM, Gutierrez A, Zhang L, Sanda T, Jette CA, Testa JR, Neuberg DS, Langenau DM, Kutok JL, Zon LI, Traver D, Fleming MD, Kanki JP, Look AT. T- lymphoblastic lymphoma cells express high levels of BCL2, S1P1, and ICAM1, leading to a blockade of tumor cell intravasation. Cancer Cell. 2010 Oct 19;18(4):353–66.

Inohara N and Nunez G. Genes with homology to mammalian apoptosis regulators identified in zebrafish. Cell Death Differ. 2000 May;7(5):509–10.

Jette CA, Flanagan AM, Ryan J, Pyati UJ, Carbonneau S, Stewart RA, Langenau DM, Look AT, Letai A. BIM and other BCL- 2 family proteins exhibit cross-species conservation of function between zebrafish and mammals. Cell Death Differ. 2008 Jun;15(6):1063–72.

Kratz E, Eimon PM, Mukhyala K, Stern H, Zha J, Strasser A, Hart R, Ashkenazi A. Functional characterization of the Bcl-2 gene family in the zebrafish. Cell Death Differ. 2006 Oct;13(10):1631–40.

Kwan TT, Liang R, Verfaillie CM, Ekker SC, Chan LC, Lin S, Leung AY. Regulation of primitive hematopoiesis in zebrafish embryos by the death receptor gene. Exp Hematol. 2006 Jan;34(1):27-34.

McNeill MS, Paulsen J, Bonde G, Burnight E, Hsu MY, Cornell RA. Cell death of melanophores in zebrafish trpm7 mutant embryos depends on melanin synthesis. J Invest Dermatol. 2007 Aug;127(8):2020–30.

Nowak M, Koster¨ C, Hammerschmidt M. Perp is required for tissue-specific cell survival during zebrafish development.

Cell Death Differ. 2005 Jan;12(1):52–64.

Peri F, Nusslein¨-Volhard C. Live imaging of neuronal degradation by microglia reveals a role for v0-ATPase a1 in phagosomal fusion in vivo. Cell. 2008 May 30;133(5):916–27.

Reyes R, Haendel M, Grant D, Melancon E, Eisen JS. Slow degeneration of zebrafish Rohon-Beard neurons during programmed cell death. Dev Dyn. 2004 Jan;229(1):30–41.

Robu ME, Larson JD, Nasevicius A, Beiraghi S, Brenner C, Farber SA, Ekker SC. p53 activation by knockdown technologies.

PLoS Genet. 2007 May 25;3(5):e78.

Santoro MM, Samuel T, Mitchell T, Reed JC, Stainier DY. Birc2 (cIap1) regulates endothelial cell integrity and blood vessel homeostasis. Nat Genet. 2007 Nov;39(11):1397–402.

Sidi S, Sanda T, Kennedy RD, Hagen AT, Jette CA, Hoffmans R, Pascual J, Imamura S, Kishi S, Amatruda JF, Kanki JP, Green DR, D’Andrea AA, Look AT. Chk1 suppresses a caspase- 2 apoptotic response to DNA damage that bypasses p53, Bcl- 2, and caspase-3. Cell. 2008 May 30;133(5):864–77.

Swaim LE, Connolly LE, Volkman HE, Humbert O, Born DE, Ramakrishnan L. Mycobacterium marinum infection of adult zebrafish causes caseating granulomatous tuberculosis and is moderated by adaptive immunity. Infect Immun. 2006 Nov;74(11):6108–17.