potential downstream of various stimuli. Remarkably, loss-of-function experiments in zebrafish embryos using morpholinos targeted against hdr, otr, or fadd did not cause significant developmental defects, indicating that the extrinsic apoptotic pathway is not critical for early zebrafish development. This is similar to blockade of the intrinsic apoptotic pathway by injecting mRNA encoding Bcl-XL or Bcl-2, which has no obvious effect on embryogenesis. Thus apoptotic pathways in zebrafish embryos may be necessary only to kill cells after severe stress or to fine-tune the numbers of cells in a specific lineage.

3.3. Chk-1 suppressed apoptosis

In a morpholino screen to discover factors for which knockdown would restore DNA-damage induced apoptosis to p53 mutant zebrafish, Sidi and colleagues (2008) elucidated a novel caspase-2 dependent apoptotic mechanism functioning downstream of Chk-1 inhibition that bypasses p53 and key components of both the intrinsic and extrinsic apoptotic pathways. This study highlighted the advantage of rapid analysis of loss of function for a panel of genes in the zebrafish embryo using morpholinos. Checkpoint kinases were quickly and systematically knocked down in a smallscale screen for modifiers of absent cell death in p53

mutant embryos. Furthermore, this group was able to examine epistatic interactions with Chk-1 inhibition (Figure 36-3) through Bcl-2 over-expression and assays for both Caspase-3 activation downstream (of which there was none) and caspase-2 activation (which became apparent only after Chk-1 knockdown). The analyses were extended into human cell culture, where Chk-1 shRNA studies in five independent p53-mutant or null cell lines confirmed the relevance of the new apoptotic pathway to human cancer cells. Because at least half of all human tumors are mutant for p53, Chk- 1 inactivation represents a novel and potentially powerful approach to sensitize p53-mutant tumor cells to the apoptotic effects of DNA-damage inducing agents, such as radiotherapy. This study bolsters use of the zebrafish system for rapid analysis of cell death pathways that can subsequently be manipulated in human tumor cells.

3.4. Anoikis

Documentation of anoikis in zebrafish comes from work by three different laboratories showing that both endothelial cells and skin keratinocytes undergo apoptosis after abnormal detachment from their extracellular matrix. The first study showed that integrin-linked kinase (Ilk) loss-of-function in zebrafish embryos causes

a loss of endothelial cells that mimics the mouse knockout phenotype. Loss of integrin signaling from attached cells is a primary cause of anoikis, also called detachment-induced cell death. In a mouse cell culture model, this group identified a loss of integrinmatrix attachment in endothelial cells as a direct cause of apoptosis. Extending these studies to transgenic zebrafish with Egfp-labeled vascular endothelial cells, they observed that Ilk-specific morpholino injection caused a reduction in the number of endothelial cells in the trunk of the embryo. Although anoikis was not specifically discussed as the cause of apoptosis in this model, this study raises the intriguing possibility that matrix attachments are crucial for cellular survival in the developing zebrafish embryo.

It was recently shown that the zebrafish Birc2 protein also acts during development to promote endothelial cell integrity and survival. As a cellular inhibitor of apoptosis (cIap), Birc2 associates with Tnfr signaling components to prevent downstream activation of extrinsic apoptosis pathways through caspase-8. Nuclear Factor kappa B (NF-κB) signaling was known to be important for vascular integrity in other contexts, and this group established that zebrafish Birc2 likely functions through NF-κB by rescuing birc2 mutant fish with the NF-κB– activating protein NEMO. Taken together, these results indicate that Birc2 may be a prosurvival factor acting downstream of external cues, such as integrin signaling, to maintain vascular integrity and endothelial cell survival.

Anoikis mechanisms also seem to be active in keratinocytes, the primary cell type found in the epidermis. Nowak and colleagues (2005) showed that the p53 signaling factor Perp functions as an antiapoptotic factor to maintain survival in cells undergoing transient developmental detachment from the underlying basement membrane. Perp morphants displayed extensive apoptosis in this tissue layer, and it is quite possible that the loss of integrin signaling during detachment triggered anoikis in this system. Future studies such as highresolution confocal time-lapse microscopy with ilk morphants, birc2 mutants, and perp morphants can further address whether anoikis-like mechanisms are truly acting to kill cells in the contexts of endothelial cell and keratinocyte detachment.

3.5. Autophagy

Recent work from Feng and colleagues has described the first evidence of autophagy in zebrafish. While analyzing adult fish with thymocytes over-expressing the oncogenes Myc and Bcl-2, these researchers dis-

Figure 36-4. Autophagy in zebrafish. Electron micrograph of one representative thymocyte in a rag2:egfp-bcl-2/rag2:myc transgenic adult zebra fish. Note the double-membraned structures denoted by arrows. These structures contain mitochondria and cytoplasm, a hallmark of autophagy. Image courtesy of Dr. Hui Feng.

covered a “small cell” phenotype. On transmission electron microscopy (TEM) analysis, they found that these cells had obvious double-membraned structures, which are rarely observed in normal cells (Figure 36-4). These double-membraned structures contained cytosol or organelles such as mitochondria, typical features of autophagosomes. Furthermore, the autophagy marker LC-3 was clearly cleaved in the Myc/Bcl-2 thymocytes, another indication of autophagosome formation. These studies establish the existence of autophagic mechanisms in zebrafish placed under specific oncogenic stresses. However, the autophagy in this context seems to be serving a prosurvival role rather than a pro-death role, which is an emerging theme in tumor cell biology. Future studies are needed to determine whether autophagy plays any role during normal zebrafish development in either cell survival or cell death.

3.6. Necrosis

Necrotic cell death in zebrafish embryos can be distinguished from apoptosis by determining that the dying cells are TUNEL-negative, cannot be rescued by a pan-Caspase inhibitor, and display membrane rupture in electron micrographs. McNeill and colleagues