The Mitochondrial Import Gene Tomm22 Is Specifically Required for Hepatocyte Survival and Provides a Liver Regeneration Model

The mitochondrial import gene tomm22 is specifically required for hepatocyte survival and provides a liver regeneration model

Silvia Curado1,*, Elke A. Ober2, Susan Walsh3, Paulina Cortes-Hernandez4, Heather Verkade5, Carla M. Koehler3 and Didier Y. R. Stainier1,*

SUMMARY Understanding liver development should lead to greater insights into liver diseases and improve therapeutic strategies. In a forward genetic screen for genes regulating liver development in zebrafish, we identified a mutant – oliver – that exhibits liver-specific defects. In oliver mutants, the liver is specified, bile ducts form and hepatocytes differentiate. However, the hepatocytes die shortly after their differentiation, and thus the resulting mutant liver consists mainly of biliary tissue. We identified a mutation in the gene encoding translocase of the outer mitochondrial membrane 22 (Tomm22) as responsible for this phenotype. Mutations in tomm genes have been associated with mitochondrial dysfunction, but most studies on the effect of defective mitochondrial protein translocation have been carried out in cultured cells or unicellular organisms. Therefore, the tomm22 mutant represents an important vertebrate genetic model to study mitochondrial biology and hepatic mitochondrial diseases. We further found that the temporary knockdown of Tomm22 levels by morpholino antisense oligonucleotides causes a specific hepatocyte degeneration phenotype that is reversible: new hepatocytes repopulate the liver as Tomm22 recovers to wild-type levels. The specificity and reversibility of hepatocyte ablation

DMM after temporary knockdown of Tomm22 provides an additional model to study liver regeneration, under conditions where most hepatocytes have died. We used this regeneration model to analyze the signaling commonalities between hepatocyte development and regeneration.

INTRODUCTION Analyzing organ regeneration can provide important insights into The liver is a crucial organ with a central role in metabolic therapeutics for pathological conditions that can benefit from tissue homeostasis: it is responsible for the metabolism, storage and repair. For example, a better understanding of liver regeneration redistribution of nutrients and it also removes waste and should enable the development of therapeutic strategies to promote xenobiotics by metabolic conversion and biliary excretion (for a endogenous liver regeneration in patients with liver diseases and, in review, see Saxena et al., 2003). Reduced liver function carries severe addition, help to increase the number of liver transplantations using consequences and, if not reversed, can be a primary cause of death. partial livers of living donors. As the main detoxifying organ of the A thorough understanding of liver development should provide a body, the liver is likely to be injured by ingested toxins. This organ valuable basis for the comprehension of disease onset and has, however, a remarkable ability to regenerate in response to injury progression, and help design successful therapeutic approaches for caused by toxic exposure, partial hepatectomy (PHx) or virus liver pathologies. Several studies in the mouse, chick, zebrafish infection (for reviews, see Fausto et al., 1995; Michalopoulos and Disease Models & Mechanisms (Danio rerio), frog (Xenopus laevis) and medaka (Oryzia latipes) DeFrances, 1997). The mechanisms responsible for liver mass have contributed to our understanding of this process and have recovery seem to be highly dependent on the form of injury. After made use of reverse and forward genetic approaches, as well as most forms of injury, including PHx, the regenerative process relies embryological and in vitro studies (for reviews, see Zhao and on the replication of the remaining hepatocytes, whereas after acute Duncan, 2005; Zaret and Grompe, 2008; Nakamura and Nishina, liver failure, a common clinical presentation of liver disease in 2009). However, much is yet to be uncovered regarding the cellular humans, the liver repopulation process appears to involve the and molecular mechanisms regulating liver development. differentiation of intrahepatic progenitor cells (for a review, see Fausto et al., 2006). Despite intensive research in the last few decades, and although the process of liver regeneration has been described extensively, the molecular mechanisms underlying this phenomenon 1 Department of Biochemistry and Biophysics, Programs in Developmental Biology, are still not clearly understood, and the exact origin of the newly Genetics and Human Genetics, Liver Center, Diabetes Center and the Cardiovascular Research Institute, University of California, San Francisco, 1550 formed cells is still debated. Fourth Street, San Francisco, CA 94158-2324, USA A number of assays have been developed to damage the liver and 2National Institute for Medical Research, Division of Developmental Biology, The subsequently study its regeneration. A 70% hepatectomy procedure Ridgeway, London, NW7 1AA, UK 3Department of Chemistry and Biochemistry, University of California, Los Angeles, (PHx resection) is commonly used in rodents to study this process Los Angeles, CA 90095, USA (for a review, see Martins et al., 2008), and has also been applied 4Section of Molecular and Cellular Biology, University of California, Davis, Davis, CA successfully in zebrafish (Sadler et al., 2007; Goessling et al., 2008). 95616, USA This approach is, however, quite laborious and time consuming, and 5School of Biological Sciences, Monash University, Clayton, VIC 3800, Australia *Authors for correspondence ([email protected]; thus is mostly applicable to a limited number of animals and mainly [email protected]) used to uncover the role of genes that have already been identified.

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As an alternative approach, we have developed a genetic/ pharmacological protocol to ablate hepatocytes specifically and in an inducible manner (Curado et al., 2007; Curado et al., 2008). This assay relies on the liver-specific expression of the bacterial nitroreductase (NTR) enzyme, which converts the non-toxic metronidazole (Mtz) into a noxious substance, ablating exclusively the cells that express NTR. The design of the NTR-Mtz system makes it suitable to damage a large number of larvae and it is therefore compatible with forward genetic and pharmacological screens. However, the NTR-Mtz system does not seem to be as efficient in ablating hepatocytes compared with other cell types, such as cardiomyocytes or pancreatic  cells (Curado et al., 2007; Pisharath et al., 2007), possibly because Mtz is being metabolized by the hepatocytes themselves, thereby lowering its effective concentration. Here, we report a surprisingly specific role for the mitochondrial import gene tomm22 in hepatocyte survival. In addition, we use this finding to generate an alternative model of liver regeneration: the transient knockdown of Tomm22 results in hepatocyte ablation followed by recovery. Using this model, we identify a role for Wnt2b and fibroblast growth factor (Fgf) signaling in hepatocyte regeneration.

DMM RESULTS oliver mutants show a liver-specific phenotype olivers469 (olis469) was identified in a forward genetic screen for genes regulating the development of endodermal organs, such as the liver, pancreas and gut (Ober et al., 2006). Screening the progeny of N- ethyl-N-nitrosourea (ENU)-mutagenized Tg(XlEef1a1:GFP)s854 zebrafish, which express green fluorescent protein (GFP) throughout oliver the developing endoderm (Field et al., 2003), led to the identification Fig. 1. mutants show a liver-specific phenotype. (A)Bright-field images of 6-dpf Tg(–2.8fabp10:EGFP)as3 WT (top) and olis469 mutant (bottom) of the recessive mutant olis469, which exhibits a smaller liver larvae combined with fluorescence to reveal their liver (arrows). By 6 dpf, and compared with wild-type (WT) siblings. We further examined the compared with their WT siblings, olis469 mutants (bottom) exhibit an o-shaped s469 as3 liver phenotype of oli mutants using the Tg(–2.8fabp10:EGFP) liver of reduced size (arrow) in an otherwise phenotypically WT body. line (Her et al., 2003), which expresses enhanced GFP (EGFP) (B,C)Confocal images of 5-dpf Tg(–2.8fabp10:EGFP)as3 WT (B) and olis469 mutant specifically in differentiated hepatocytes, combining bright-field and (C) livers stained with phalloidin (red) and TO-PRO (blue). EGFP expression fluorescence imaging. At 6 days post-fertilization (dpf), the mutant (green) in hepatocytes reveals a well-organized structure in the WT liver, s469 larvae (Fig. 1A) exhibit an o-shaped liver (o-liver) of drastically contrasting with the disarray observed in oli mutants. (D,E)Confocal images gz12 s469 reduced size compared with their WT siblings (Fig. 1A). At the of 5-dpf Tg(fabp10:RFP) WT (D) and oli mutant (E) livers stained for Disease Models & Mechanisms s469 cadherin (green) and TO-PRO (blue). The cells surrounded by the few morphological level, only the liver appears to be affected in oli hepatocytes (red) that are detected in s469 mutant livers express cadherin s469 oli mutants; oli mutant larvae survive until 9-11 dpf without showing (green) at levels that, at this stage in WT livers, are characteristic of cells of the any other obvious phenotype. At this stage, most mutant larvae have intrahepatic biliary tree (D, arrows). Bars, 20m. inflated their swim bladder although they are thinner than their WT siblings, probably owing to the lack of a functional liver. To gain further insight into the liver defect, we stained 5-dpf cells and hepatocytes is also altered, with a much higher number Tg(–2.8fabp10:EGFP)as3; olis469 larvae with phalloidin and the of bile duct cells compared with hepatocytes. nuclear marker TO-PRO. Whereas, at this stage, WT livers consist To further understand the origin of the olis469 mutant liver of multiple hepatocytes that are well organized in a sheet-like phenotype, we examined the hepatic structure at earlier stages. At structure (Fig. 1B), olis469 mutant livers show a very distinct hepatic 4 dpf, both WT and olis469 mutant livers exhibit a mesh-like structure, where only a few hepatocytes can be detected (Fig. 1C). intrahepatic biliary duct structure, highlighted by cadherin and Confocal imaging of 5-dpf mutant larvae expressing red fluorescent 2F11 staining (Fig. 2A,B). Twenty-four hours later, in olis469 mutant protein (RFP) in hepatocytes [Tg(fabp10:RFP)gz12] (Farooq et al., livers, this biliary duct structure forms a core of cadherin/2F11- 2008), stained with an antibody against cadherin and with TO-PRO, positive cells surrounded by a few remaining hepatocytes (Fig. 2D), revealed that the cells that were surrounded by only a few as opposed to the WT liver structure, where the hepatic biliary hepatocytes (Fig. 1E) expressed high cadherin levels, which, at this ducts and hepatocytes intermix (Fig. 2C). stage in WT livers, are only present in the biliary duct cells (Fig. 1D). Moreover, analysis of the hepatic structure in olis469 mutants The oliver mutant phenotype is caused by hepatocyte apoptosis clearly shows that, not only is the overall size and organization of Both bile duct cells and hepatocytes derive from hepatoblasts, the liver affected in these mutants, but the ratio between bile duct which in turn originate from the primary liver bud endoderm

Disease Models & Mechanisms 487 RESEARCH ARTICLE Tomm22 is specifically required for hepatocyte survival

(TdT-mediated dUTP nick-end labeling). Whereas no TUNEL staining was detected in WT livers (Fig. 2E), we observed many TUNEL-positive hepatocytes [marked by Tg(fabp10:RFP) expression] in olis469 mutants (Fig. 2F). Confocal imaging of 4- dpf TUNEL-stained sections clearly shows that, in tissues other than the liver (such as the gut), the low number of apoptotic cells detected in olis469 mutants is similar to that observed in WT siblings (supplementary material Fig. S1A,B). In addition, using the monoclonal antibody WCL15 (Romano et al., 1998; van der Sar et al., 2004), we detected several macrophages engulfing dying hepatocytes in olis469 mutants (Fig. 2H), whereas in WT livers macrophages can only very occasionally be observed in the periphery of the liver (Fig. 2G). In addition, to assess hepatocyte differentiation, we examined the distribution of the bile salt export pump Abcb11 (Gerloff et al., 1998; Sakaguchi et al., 2008) at 4 dpf, and observed clear labeling of mutant hepatocytes with this marker (data not shown). Altogether, these results indicate that the mutant hepatocytes are specified, differentiate and then undergo apoptosis. Although, at 4 dpf, olis469 mutant livers exhibit a WT-like cellular organization with sheets of hepatocytes separated by biliary ducts, owing to hepatocyte apoptosis, their size is already much reduced compared with WT siblings. DMM tomm22 is essential for hepatocyte survival To isolate the gene disrupted by the olis469 mutation we used a standard set of simple sequence length polymorphism (SSLP) markers and placed the olis469 locus on linkage group 12. Further mapping positioned this mutation within a 140-kb genomic interval containing eight genes (supplementary material Fig. S2A). Comparative cDNA sequence analysis revealed a nucleotide change from thymine in WT fish, to cytosine in olis469 mutants, in the translocase of the outer mitochondrial membrane 22 (tomm22) gene (supplementary material Fig. S2B). This nucleotide change results in the substitution of a stop codon with an arginine residue, leading Fig. 2. The oliver mutant phenotype is caused by hepatocyte apoptosis. to a predicted mutant protein that is 47 amino acids longer than (A-D)Confocal images of 4-dpf (A,B) and 5-dpf (C,D) Tg(fabp10:RFP)gz12 WT the WT protein (supplementary material Fig. S2C). (A,C) and olis469 mutant (B,D) livers stained for cadherin (blue) and 2F11 Tomm22 is part of the multiprotein translocation complex of s469 (green). At 4 dpf, both WT and oli mutant livers exhibit a distinct the outer mitochondrial membrane (OMM) that regulates protein Disease Models & Mechanisms intrahepatic biliary tree (highlighted by the expression of cadherin and 2F11), traffic from the cytoplasm into mitochondria. Mitochondrial intercalated by hepatocytes (red). However, 24 hours later (5 dpf), the biliary tree in olis469 mutants has turned into a dense cellular mass. (E,F)Confocal import is crucial, as the majority of mitochondrial proteins are images of transverse sections of 4-dpf Tg(fabp10:RFP)gz12 WT (E) and olis469 encoded by nuclear DNA and thus need to be imported after mutant (F) livers stained for cadherin (blue) and TUNEL (green); the livers are cytosolic translation. Tomm22 acts as a central receptor and a trans outlined by dashed lines. At 4 dpf, a large number of hepatocytes (red) binding site for this translocation, as well as a docking point for undergo apoptosis in olis469 mutant livers (F), whereas no TUNEL staining can other peripheral Tomm components of the mitochondrial import be detected in the livers of their WT siblings (E). (G,H)Confocal images of 4-dpf pore (for a review, see Koehler, 2004). To date, only one zebrafish gz12 s469 Tg(fabp10:RFP) WT (G) and oli mutant (H) livers stained with the tomm22 gene has been identified. macrophage antibody WCL15 (green). As hepatocytes (red) undergo Interference with tomm22 translation (or knockdown of the apoptosis in olis469 mutant livers, multiple macrophages (green) can be detected engulfing them (H), whereas in their WT siblings, macrophages can Tomm22 protein), using 4 ng of an ATG-targeting morpholino s469 only very occasionally be detected in the periphery of the liver (G). Bars, antisense oligonucleotide (MO), accurately reproduced the oli 20m. phenotype (Fig. 3A-C). Confocal analysis of these morphants showed that their livers consisted of a mass of cadherin/2F11- positive cells surrounded by a few hepatocytes (Fig. 3E), as detected (Zhao and Duncan, 2005). There are at least two possible in olis469 mutants (Fig. 3D). These results confirm that the liver explanations for the development of the abnormal bile duct phenotype observed in olis469 mutants is caused by the disruption cell:hepatocyte ratio that is observed in olis469 mutants: of the tomm22 gene. hepatocyte-specific apoptosis or a bias of hepatoblast The tight genetic linkage, presence of a significant molecular differentiation towards the bile duct lineage. To test these lesion in the tomm22s469 coding sequence, and the tomm22 MO hypotheses, we examined the extent of apoptosis using TUNEL phenocopy indicate that a disruption in the tomm22 gene is the

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Fig. S3A). Carbonate removes proteins from the mitochondrial membranes (pellet fraction) with increasing pH, whereas soluble proteins and unimported proteins remain in the supernatant fraction. We found that, similar to the WT protein, the imported Tomm22s469 mutant protein remained associated with the membrane fraction even at the high pH of 12.5. In addition, a control of carbonate extraction in the absence of mitochondria showed that this pellet association was not simply the result of protein aggregation (data not shown). To further test that Tomm22s469 was indeed in the OMM, we performed import assays and then added trypsin to degrade the exposed proteins (supplementary material Fig. S3B). Tomm22s469, like WT Tomm22 and the yeast ortholog Tom22, was degraded in these experiments, indicating that it localizes to the OMM. Taken together, these results indicate that Tomm22s469 is correctly imported and localized to the yeast OMM, like the WT form. Thus, the lack of Tomm22s469 function in olis469 mutants is likely to be the result of an effect other than inappropriate targeting.

tomm22 is expressed highly in the endoderm We next investigated tomm22 expression in WT larvae using a tomm22-specific probe, as well as a control sense probe.

DMM Throughout 3-5 dpf, tomm22 appears to be highly expressed in the endoderm-derived organs – the liver, gut and, although not as strongly, the pancreas – as compared with other tissues (supplementary material Fig. S4A-D). This staining pattern was s469 Fig. 3. tomm22 MO injections reproduce the oli liver-specific clearly absent when using the sense control probe (supplementary phenotype. (A)Bright-field image of a 4-dpf uninjected control larva (top) and material Fig. S4E-G’). tomm22 expression was also detected in the a 4-dpf larva injected with 4 ng of tomm22 MO (bottom). (B,C)Fluorescence head, although weaker nonspecific staining in the head could also images of 4-dpf (B) and 5-dpf (C) Tg(fabp10:RFP)gz12 larvae injected with 4 ng of tomm22 MO (left) and uninjected controls (right). The 4-dpf and 5-dpf be detected in the sense control. This enriched expression in tomm22 morphants show a liver-specific phenotype: the size of the liver is endodermal organs during embryogenesis is in contrast to the low much reduced (arrows in B,C) in an otherwise WT-like body (A, bottom) level of ubiquitous expression that is seen in mature mammals compared with the uninjected controls. (D,E)Confocal images of 4-dpf (Unigene; www.ncbi.nlm.nih.gov/unigene). Tg(fabp10:RFP)gz12 livers from uninjected (D) and tomm22 MO-injected (E) animals stained for cadherin (blue) and 2F11 (green). The hepatic structure of Tomm22 transient knockdown provides an additional model to s469 the tomm22 morphants, similar to that observed in oli mutants, consists of study liver regeneration a mass of cadherin- and 2F11-positive cells surrounded by residual Injection of the tomm22 MO results in the specific apoptosis of hepatocytes (in red) (E), in contrast to the cellular organization that is observed in uninjected larvae (D). Bars, 20 m. hepatocytes, and can therefore be used as a tool to ablate Disease Models & Mechanisms  hepatocytes. Contrary to the permanent absence of Tomm22 in tomm22s469 mutant larvae, the knockdown of Tomm22 levels using cause of the olis469 mutant phenotype and, therefore, that tomm22 a tomm22 MO is transient, as this reagent is gradually depleted. plays a crucial role in hepatocyte survival. Thus, whereas the liver phenotype in tomm22s469 mutants is Like the liver, the heart has a high demand for mitochondrial irreversible, once Tomm22 levels are re-established in tomm22 function. However, tomm22s469 mutant hearts show no obvious morphants, the degenerated liver recovers its size and structure. morphological defects or function impairment (data not shown). tomm22 morphants, which show a smaller liver with a reduced number of hepatocytes at 5 dpf, show a recovering hepatic mass Tomm22s469 mutant protein is correctly inserted into the yeast at 6 dpf and, by 8 dpf, start resembling uninjected controls (Fig. OMM 4A). The ability to ablate hepatocytes specifically using a tomm22 Like the majority of the mitochondrial proteins, Tomm22 is MO, together with the reversibility of its effect, make the tomm22 encoded by a nuclear gene. Tomm22 is initially translated in the MO-based assay a useful model to study liver regeneration when cytosol, then imported into the mitochondria, and integrated in hepatocyte replication is absent or impaired. the OMM (Becker et al., 2009). To investigate whether the Although it is known that mature hepatocytes are the main phenotype observed in tomm22s469 mutants was the result of source of regenerating cells following liver damage such as PHx inappropriate targeting of the Tomm22s469 mutant protein, we (for a review, see Fausto et al., 2006), in conditions where there is used a yeast in vitro import system (Hwang et al., 2007). low or no mature hepatocyte replication, the source of new hepatic Radiolabeled WT or Tomm22s469 mutant protein was imported cells is still highly debated. Following tomm22 MO-induced into WT yeast mitochondria and the import reaction was split ablation, we followed cellular proliferation at 3, 4 and 5 dpf and and carbonate-extracted at various pHs (supplementary material observed a higher number of proliferating [phospho-histone H3

Disease Models & Mechanisms 489 RESEARCH ARTICLE Tomm22 is specifically required for hepatocyte survival

Fig. 4. tomm22 MO provides a model to study liver regeneration and uncovers an involvement of the Wnt2b signaling pathway. (A)Fluorescence images of 5-, 6- and 8-dpf (left, middle and right images, respectively) Tg(fabp10:RFP)gz12 larvae injected with 4 ng of tomm22 MO (left side of each image) and uninjected control larvae (right side of each image). At 5 dpf, tomm22 morphants show a smaller liver (arrow); as the tomm22 MO is diluted and Tomm22 levels recover, the tomm22 morphant livers start recovering their size and structure, which is already obvious at 6 dpf, and are comparable to uninjected controls at 8 dpf. (B)Confocal images of the livers of Tg(fabp10:RFP)gz12 WT (top) and tomm22 MO-injected (bottom) larvae at 3, 4 and 5 dpf (left, middle and right panels, respectively) stained for PH3 (green) and Alcam (blue); hepatocytes are stained red. Cellular proliferation occurs at a higher rate in post-ablated livers compared with WT livers. Bars, 20m. (C)Comparison of hepatic recovery post-tomm22 MO-mediated ablation in the WT (left bar, n41) versus wnt2bb–/– (right bar, n42) background. tomm22 morphants, which at 4 dpf showed severe hepatic ablation, were allowed to recover and at 6 dpf their liver recovery was classified as (1) ‘no’ change in liver size, (2) ‘moderate’ recovery of hepatic volume, or (3) ‘full’ recovery, i.e. comparable to that observed at 6 dpf in most WT larvae injected with 4 ng of tomm22 MO. In the absence of wnt2bb function, ‘full’ recovery (blue) decreased from 67.1% to 1.9%. Conversely, ‘no’ recovery (yellow) increased from 5.2% in WT larvae to 59.5% in wnt2bb–/– larvae. ‘Moderate’ recovery changed from 27.7%

DMM in WT larvae to 38.6% in wnt2bb–/– larvae. (D)Comparison of hepatic recovery post-tomm22 MO-mediated ablation in larvae that were allowed to recover in carrier solution (DMSO; left bar, n73) compared with in the presence of 5M of the Wnt–-catenin pathway inhibitor IWR-1 (right bar, n82). Recovery rates were classified as in C. In the presence of the Wnt–-catenin pathway inhibitor IWR-1, ‘full’ recovery decreased compared with DMSO (from 27.5% to 7%), whereas the ‘no’ recovery rate increased from 45% (DMSO) to 62% (5M IWR-1). No clear difference was observed in the ‘moderate’ recovery category (27.5% to 31%). (E-F’) Expression of wnt2bb in 4-dpf uninjected (E,E’) and tomm22 MO-injected (F,F’) larvae. E and F are dorsal views; E’ and F’ are lateral views. wnt2bb hepatic expression appeared to be upregulated in many 4-dpf tomm22 morphants (n9/17) (F, F’) compared with uninjected larvae (n0/17) (E, E’) (similar numbers were observed at 5 dpf). Disease Models & Mechanisms

(PH3)-positive] cells in recovering tomm22 morphants compared et al., 2008). However, these studies have been carried out after with WT livers, and found that the proliferating cells were also PHx. To investigate whether the Wnt–-catenin signaling pathway Alcam positive (Fig. 4B). This result suggests that, in conditions is also involved in the regeneration process after severe hepatocyte where mature hepatocyte replication is absent or impaired, cells ablation, we used the tomm22 MO-based regeneration assay in the expressing Alcam, which at this stage in WT livers corresponds to wnt2bbs403 mutant background (Ober et al., 2006). biliary epithelial cells (Sakaguchi et al., 2008), may be responsible tomm22 MO-injected larvae (WT, wnt2bb+/– and wnt2bb–/–), for replenishing the liver with new hepatocytes. exhibiting a small liver at 4 dpf, were analyzed at 6 dpf for liver repopulation. At this stage, under control conditions, the liver mass The Wnt2b signaling pathway positively regulates hepatocyte of tomm22 morphants has recovered significantly (Fig. 4A). Hepatic regeneration recovery was classified into three categories: ‘full’ recovery The Wnt–-catenin signaling pathway has been implicated in the (comparable to recovery in the WT background), ‘moderate’ regeneration process of a number of tissues, including the liver recovery (new hepatocytes formed but to a lesser extent than in (Monga et al., 2001; Sodhi et al., 2005; Tan et al., 2006; Goessling ‘full’ recovery) and ‘no’ recovery (the liver size remained the same

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as at 4 dpf). To investigate the correlation between liver recovery import pore (GIP) and is formed by a complex of proteins: and wnt2bb function, larvae were sorted at 6 dpf into one of the Tomm22, Tomm20, Tomm70, Tomm40, Tomm5, Tomm6 and three categories and then genotyped for the wnt2bbs403 mutation. Tomm7. Tomm22 is a well-conserved translocase that, in yeast, We observed that, in the absence of wnt2bb function, ‘full’ recovery has been shown to function as an essential receptor for the was rarely observed (67.1% in non-mutants, 1.9% in wnt2bb–/– proteins that are recognized initially by Tomm20 and Tomm70. mutants), and conversely, ‘no’ recovery increased (5.2% in non- Tomm22 then transfers these proteins to the import pore and, as mutants, 59.5% in wnt2bb–/– mutants) (Fig. 4C). they pass through the Tomm40 channel, they interact with the wnt2bb mutant larvae exhibit early liver development defects, Tomm22 intermembrane space domain. In addition, Tomm22 is however, liver size becomes comparable to WT by 4 dpf (the time essential for stabilizing the integrity of the import pore (for a point when tomm22 MO-injected larvae were selected, based on review, see Pfanner and Wiedemann, 2002). Limited loss-of- their liver size, to follow hepatic regeneration). Nevertheless, to function studies of translocases carried out in multicellular further test the involvement of the Wnt–-catenin signaling organisms have revealed defects in multiple tissues. In zebrafish, pathway in this hepatocyte recovery model, we exposed tomm22 timm50 morphants exhibit multiple developmental defects morphants to the Wnt–-catenin inhibitor IWR-1 (Chen et al., including dysmorphic hearts, neurodegeneration, reduced 2009), starting at 4 dpf. Inhibition of the Wnt–-catenin signaling motility and, eventually, lethality at 4 dpf (Guo et al., 2004); pathway using IWR-1 also resulted in the inhibition of hepatic knockdown of three different translocases of the inner regeneration. In the presence of 5 M of IWR-1, the ‘full’ recovery mitochondrial membrane (Timm) in Caenorhabditis elegans was rate decreased (27.5% in controls, 7% in 5 M IWR-1-treated shown to result in several developmental defects, affecting mostly larvae), whereas the ‘no’ recovery rate increased (45% in controls, cell size, and partial lethality (Curran et al., 2004); and Timm23 62% in 5 M IWR-1-treated larvae) (Fig. 4D). These results indicate knockout mice show a neurological phenotype and reduced life a role for the Wnt–-catenin signaling pathway in hepatic span (Ahting et al., 2009). Our finding that the absence of regeneration post-tomm2 MO-induced hepatocyte ablation and are Tomm22, a protein thought to be a major player in the general

DMM consistent with the recovery results observed in the absence of import process common to all mitochondria, results in the wnt2bb function. To further investigate the role of Wnt–-catenin specific apoptosis of hepatocytes is unexpected and intriguing. signaling during liver regeneration, we examined tomm22 There can be several explanations for the tissue-specific morphants at 4 and 5 dpf, and observed an upregulation of wnt2bb phenotype in tomm22s469 mutants. We observed that this gene is expression in their hepatic region compared with controls (Fig. 4E- expressed highly in the developing endodermal organs – the liver, F’). Thus, wnt2bb expression appears to be induced following gut and, although at a lower level, the pancreas – and thus there hepatocyte apoptosis in tomm22 morphants. Similar to the results may be a higher requirement for Tomm22 in these organs. Although obtained using the Wnt–-catenin inhibitor IWR-1, we observed no obvious phenotype was detected in the gut of tomm22 mutants a consistent inhibitory effect on hepatic regeneration when or morphants, some of these embryos showed a slightly smaller recovering larvae were exposed to the Fgf receptor (FgfR) inhibitor pancreas and/or lower Tg(XlEef1a1:GFP)s854 pancreatic expression. SU5402 (supplementary material Fig. S5). However, when using the Previous work has reported that, across 14 different adult mouse bone morphogenetic protein (Bmp) signaling pathway inhibitor tissues, the heart has the highest mitochondrial density, whereas dorsomorphin (Yu et al., 2008), we did not observe an obvious effect the liver showed a third of that density and less than many other on hepatic regeneration (data not shown). organs including the large and small intestine, kidney, skeletal muscle and neural tissues (Pagliarini et al., 2008). These findings DISCUSSION point to a qualitative, rather than quantitative, difference in Disease Models & Mechanisms Our study revealed an unexpectedly specific requirement for mitochondrial tissue requirement as the cause of the tissue-specific Tomm22 in hepatocyte survival. In tomm22 mutants, the liver is phenotype in tomm22s469 mutants. initially specified, bile ducts form and hepatocytes differentiate, but Indeed, mitochondria perform a variety of different functions hepatocytes die shortly thereafter, leading to a small liver consisting whose relevance depends on the specific tissue, or even on their mainly of biliary tissue. specific location within the cell (Kuznetsov et al., 2004). Pagliarini Mitochondria provide eukaryotic cells with energy and play et al. (Pagliarini et al., 2008) also assessed the relative abundance other crucial roles in cell signaling, the metabolism of amino acids of each mitochondrial protein across 14 different murine tissues and lipids, the biosynthesis of heme and iron-sulfur clusters, and and revealed that approximately only a third of the 1098 apoptosis. In humans, only 13 mitochondrial proteins are encoded mitochondrial proteins are present across all the sampled tissues, by the mitochondrial genome, the remaining roughly 99% are whereas most of the mitochondrial proteins show some tissue encoded by nuclear genes and, thus after their synthesis on specificity. The finding that liver mitochondria contain cytosolic ribosomes, need to be imported into the four different mitochondrial proteins that are different from the ones present in mitochondrial compartments (outer membrane, inner membrane, other tissues can help explain the specificity of the phenotype intermembrane space and matrix) (Bolender et al., 2008). Most observed in tomm22 mutants. Thus, we hypothesize that Tomm22 mitochondrial proteins are translocated across the OMM using is essential for the import of a specific protein or set of proteins the TOM (translocase of the outer membrane) complex, which required for a particular mitochondrial function that is necessary is the entry gate of the mitochondria (Endo et al., 2003; Koehler, for hepatocyte survival. Of course, we cannot exclude the possibility 2004; Rehling et al., 2004; Neupert and Herrmann, 2007). Multiple of functional redundancy in non-hepatic tissues by (1) an alternative studies on mitochondrial import, carried out mainly in yeast or bypass mechanism that is independent of the TOM complex [such in vitro, have shown that the TOM complex includes the general as the one shown for cytochrome P450 import in yeast

Disease Models & Mechanisms 491 RESEARCH ARTICLE Tomm22 is specifically required for hepatocyte survival

(Anandatheerthavarada et al., 2009)], or (2) the existence of a second or other Alcam-positive cells. Lineage experiments with tomm22 gene in the zebrafish genome, or of another gene that appropriate Cre-expressing lines will be required to test this functions like tomm22. hypothesis rigorously. In previous work, Wnt2b signaling has been The specific hepatocyte apoptosis observed in tomm22s469 shown to positively regulate liver development (Ober et al., 2006; mutants and tomm22 MO-injected larvae; the regeneration of McLin et al., 2007; Tan et al., 2008). Liver specification is initially hepatocytes once the Tomm22 levels are re-established; and the impaired in zebrafish wnt2bb mutants, although the liver high expression levels of tomm22 detected in the endodermal eventually forms in most of these animals (Ober et al., 2006). Using tissues, such as the liver, point to a specific role for tomm22. Most our assay, we observed that Wnt2b signaling plays a role in the studies on mitochondrial protein import in multicellular regeneration of the ablated hepatic tissue, suggesting that it may organisms have been carried out in vitro or ex vivo, and only a also play a role in a progenitor-dependent liver regeneration few have addressed the effect of the loss of function of a process. The decrease in liver regeneration rate in the presence mitochondrial import protein in vivo. To our knowledge, the of a Wnt–-catenin pathway inhibitor (IWR-1), as well as the finding that the absence of vertebrate Tomm22 function appears upregulation of wnt2bb in the liver region in post-ablation to affect mainly the maturing hepatocytes has not been described recovering larvae, provides additional support for the involvement before. of this pathway in the hepatic regeneration process following tomm22 MO-mediated hepatocyte ablation. Tomm22 liver regeneration model introduces another application for morpholino-based assays Development and regeneration In this study, we showed that the temporary knockdown of Several aspects of tissue development are thought to be Tomm22 levels through the use of tomm22 MO resulted in the recapitulated during the process of regeneration, as both specific ablation of hepatocytes followed by hepatic tissue recovery. processes involve common mechanisms such as proliferation, MOs have been mostly used in developmental studies, as a tool for differentiation, patterning and growth control. However, the

DMM reverse genetics to uncover the role of a specific target protein in extent of the similarity between the two processes remains different animal models, but also in therapeutics, targeting bacteria unclear. Whereas some studies suggest parallels between the two or viruses (Nasevicius and Ekker, 2000; Heasman, 2002; Geller, processes, as shown by the similarity of the antigenic and 2005; Deas et al., 2007). Our tomm22 MO-based assay provides a biochemical profiles of pluripotent progenitors in the liver during model to study liver regeneration, highlighting an additional development and regeneration (Zhang et al., 2008), a recent study application for the MO technology. The use of MO-based assays, reported that progenitor cells involved in muscle development such as the one described here, can be quite powerful when applied (embryonic muscle progenitors) and during regeneration (adult to larger-scale regeneration studies, where larger numbers of satellite cells) show significant differences in their genetic samples can be tested. This approach may allow the screening of requirements (Lepper et al., 2009). a large number of pharmacological substances for their potential The role of Wnt2b and Fgf signaling in the tomm22 MO to enhance or impair regeneration or, in forward genetic screens, regeneration model suggests a certain level of similarity between the for the identification of genes that modify this process. Moreover, pathways regulating liver development and regeneration. These MO-based regeneration assays may prove to be especially useful findings are in agreement with a promoting role for Wnt2b and Fgf for later developmental stages when using the emerging caged MO signaling in liver specification (Jung et al., 1999; Rossi et al., 2001; technology. In the latter approach, the MO is photoactivatable, Ober et al., 2006; Shin et al., 2007), as well as in hepatocyte which allows spatiotemporal protein downregulation (Shestopalov proliferation-based recovery post-PHx (Monga et al., 2001; Steiling Disease Models & Mechanisms et al., 2007). Alternatively, a conditional transgenic rescue of the et al., 2003; Sturm et al., 2004; Goessling et al., 2008; Kan et al., 2009). tomm22 mutation may also allow late-onset hepatocyte apoptosis Although the Bmp pathway has also been shown to be involved in followed by regeneration. liver development (Rossi et al., 2001), as well as in regeneration post- PHx (Steiling et al., 2003; Sturm et al., 2004; Sugimoto et al., 2007; Wnt2b signaling is involved in liver regeneration Kan et al., 2009) or following tetrachloride-induced injury (Nakatsuka Several studies have suggested that Wnt–-catenin signaling is et al., 2007), we observed no clear role for Bmp signaling based on involved in the regeneration of different tissues, such as the newt dorsomorphin treatments. In the future, it will be important to dissect lens (Hayashi et al., 2008), bone (Zhong et al., 2006), muscle the specific roles of these signaling pathways in various models of (Polesskaya et al., 2003) and zebrafish tail fin (Stoick-Cooper et liver regeneration. The tomm22 MO assay will be a powerful tool in al., 2007). The Wnt–-catenin pathway has also been shown to such studies, as it is easily scalable. be activated during liver regeneration after PHx in rodents (Monga et al., 2001) and zebrafish (Goessling et al., 2008), and METHODS downregulation of -catenin has been shown to partially inhibit Zebrafish strains this process in the rat and mouse (Sodhi et al., 2005; Tan et al., Zebrafish embryos, larvae and adult fish were raised under standard 2006). In contrast to PHx, the hepatic recovery assay that we laboratory conditions at 28°C (Westerfield, 2000). We used the developed and used in this study is likely to be progenitor cell following mutant and transgenic lines: wnt2bbs403 (Ober et al., dependent as, after hepatocyte ablation, only very few hepatocytes 2006), Tg(fabp10:RFP, ela3l:EGFP)gz12 (Farooq et al., 2008) [for remain. Our proliferation assays suggest that, in the tomm22 MO simplicity, this line is referred to as Tg(fabp10:RFP)gz12 in this hepatocyte ablation model, the newly formed hepatocytes do not manuscript], Tg(–2.8fabp10:EGFP)as3 (Her et al., 2003) and derive from other hepatocytes but rather from biliary duct cells Tg(XlEef1a1:GFP)s854 (Field et al., 2003).

492 dmm.biologists.org Tomm22 is specifically required for hepatocyte survival RESEARCH ARTICLE

Sectioning, immunohistochemistry and microscopy Larvae were fixed overnight at 4°C with 3% formaldehyde and TRANSLATIONAL IMPACT then immunostained in whole mounts or following sectioning. Clinical issue For sectioning, larvae were embedded in low-melting agarose and Liver disease results from intrinsic causes, such as mitochondrial defects or cut into 200-m sections with a vibratome. Antibody staining was cancer, or from exposure to external agents such as chemicals, toxins or carried out in PBST (0.1% Triton X-100, 4% BSA, 0.02% NaN3 in viruses. Although a damaged liver can regenerate, acute liver damage and PBS, pH 7.3). We used the following antibodies: polyclonal failure is normally treated by organ transplantation, which is hampered by a antibody against cadherin (rabbit, 1:1000; Sigma); 2F11 severe shortage of suitable donors and, even when successful, reduces patient life span significantly. To help improve transplant procedures, and ultimately to monoclonal antibody (mouse, 1:400; a generous gift from Julian provide a source of healthy hepatic tissue, the process by which the liver Lewis, Cancer Research, UK), 2F11 is used as a marker of the regenerates, and how this process can be enhanced, needs to be better intrahepatic biliary duct in the zebrafish liver and for secretory understood. cells in the zebrafish gut (Crosnier et al., 2005); WCL15 Results monoclonal antibody (mouse, 1:100; a generous gift from Jan This paper describes a zebrafish model for liver regeneration. The authors used Rombout, University of Tuscia, Italy); Zn8 monoclonal antibody a forward genetic screen in which they chemically mutagenized zebrafish against Alcam (mouse, 1:10; Developmental Studies Hybridoma adult males and then looked for liver defects in their F3 progeny – this screen Bank, University of Iowa); anti-phospho-histone H3 (PH3) led to the identification of a mutant they called oliver. oliver mutants have an antibody (rabbit, 1:100; Upstate); and fluorescently conjugated o-shaped liver (o-liver) of much reduced size owing to the depletion of most of the hepatocytes. oliver maps to the tomm22 gene, which encodes a translocase secondary antibodies from Molecular Probes (1:200). For F-actin of the outer mitochondrial membrane, thought to play an important role in or nuclear staining, whole larvae and sections were stained with protein import into mitochondria. Morpholino injections, in which modified rhodamine phalloidin (1:100, Molecular Probes) or TO-PRO 3 oligonucleotides (morpholinos) directed against tomm22 mRNA inhibited (1:5000, Molecular Probes), respectively. Images were acquired tomm22 translation, showed that knockdown of Tomm22 protein resulted in with a Zeiss 510 confocal or Zeiss Lumar microscope and the same phenotype as that found in oliver mutants, thus confirming that DMM prepared for publication using Adobe Photoshop and Illustrator. tomm22 is required for hepatocyte survival. Importantly, as protein knockdown by morpholino only lasts until the morpholino is used up, the authors could show that when Tomm22 levels recovered, the liver mass also recovered, TUNEL cell death assay suggesting that liver damage caused by mitochondrial dysfunction can be The TUNEL cell death assay was performed using an In Situ Cell reversible. Loss of the Wnt2b signaling pathway, which is required for liver Death Detection Kit, Fluorescein (Roche; cat. no. 11 684 795 001). development, inhibited the regeneration process in tomm22 morpholino- After fixation of zebrafish larvae in 3% formaldehyde and injected embryos, indicating some overlap in the control mechanisms sectioning, transverse sections of the liver were pre-incubated in between normal development and regeneration. PBST and then labeled with the TUNEL kit for 1 hour at 37°C. Implications and future directions Sections were washed with PBT (0.1% Triton X-100 in PBS, pH The ease of producing and screening large numbers of zebrafish makes the 7.3) and visualized by confocal imaging. For co-immunostaining tomm22 liver regeneration assay suitable for large-scale chemical and genetic screens for molecules or genes modulating hepatocyte regeneration in the with cadherin, sections were first incubated with primary absence of most hepatocytes. tomm22 mutant fish will also be useful for antibodies, then with TUNEL solutions, and finally with secondary identifying the lineage from which regenerating hepatocytes originate, and antibodies. provide a whole-organism model for studying hepatic mitochondrial diseases. doi:10.1242/dmm.005637 tomm22s469

Disease Models & Mechanisms Mapping and genotyping of the allele The mutation in olis469 was mapped to linkage group 12 using a set of SSLP markers. For fine mapping, 1363 mutant larvae were Purification of yeast mitochondria and in vitro import assays WT GA74 yeast mitochondria were isolated from cultures grown tested for SSLPs in the critical region, followed by isolation of cDNA at 30°C, to an OD of 2, in rich lactate medium (1% yeast extract, from olis469 larvae and siblings, and sequence analysis of candidate 600 2% tryptone, 0.05% dextrose, 2% lactic acid, 3.4 mM CaCl , 8.5 mM genes within the critical interval, including tomm22. 2 NaCl, 2.95 mM MgCl , 7.35 mM KH PO , 18.7 mM NH Cl). Detection of the tomm22s469 allele was performed by PCR 2 2 4 4 Mitochondria were isolated as described previously (Daum et al., amplification of genomic DNA using the forward 5Ј-CAGAG- Ј Ј 1982). WT or mutant Tomm22 in pCS2 (SP6), or Tom22 in GAGAGATAATCAAACAGGT-3 and reverse 5 -TGGACGT- pcGlobin2 (T7), were translated using a TnT kit (Promega) in the Ј CAGTCTATGCAGAA-3 primers, followed by digestion with presence of 35S-methionine. Radiolabeled precursors were Fnu4HI, generating digestion products of 305, 73, 17 and 120 bp incubated at 30°C with isolated mitochondria in import buffer (1 in the tomm22s469 allele, and products of 378, 17 and 120 bp in the mg/ml BSA, 0.6 M sorbitol, 150 mM KCl, 10 mM MgCl2, 2.5 mM WT. EDTA, 2 mM ATP, 2 mM NADH, 20 mM Hepes-KOH, pH 7.4) for 30 minutes. Outer membrane proteins were digested with 1 Morpholino antisense oligonucleotide design and injection g/ml trypsin for 30 minutes on ice. Trypsin was inhibited with The tomm22 morpholino oligonucleotide (tomm22 MO) was 10 g/ml of soybean trypsin inhibitor. Mitochondria were pelleted targeted against the translational site of tomm22 with the following at 14,000 ϫ g for 5 minutes at 4°C, and resuspended in SDS-PAGE sequence: 5Ј-GAGAAAGCTCCTGGATCGTAGCCAT-3Ј (Open sample buffer. For carbonate extraction, a single import reaction Biosystems). Embryos were injected with 4 ng of tomm22 MO at was divided into four tubes. Mitochondria were collected by the one-cell stage. spinning at 8000 ϫ g for 5 minutes at 4°C and then resuspended

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in 0.1 M sodium carbonate, at various pHs, or in Thorner buffer Alexander, J., Stainier, D. Y. and Yelon, D. (1998). Screening mosaic F1 females for (10% glycerol, 8 M urea, 5% SDS, 40 mM Tris, pH 6.8, 4 mg/ml mutations affecting zebrafish heart induction and patterning. Dev. Genet. 22, 288- 299. Bromophenol Blue, 5% -mercaptoethanol). After 30 minutes on Anandatheerthavarada, H. K., Sepuri, N. B. and Avadhani, N. G. (2009). ice, the mitochondria were pelleted at 14,000 ϫ g for 10 minutes Mitochondrial targeting of cytochrome P450 proteins containing NH2-terminal at 4°C. The pellet containing the membrane fraction was chimeric signals involves an unusual TOM20/TOM22 bypass mechanism. J. Biol. Chem. 284, 17352-17363. resuspended in Thorner buffer, and the supernatants were Becker, T., Gebert, M., Pfanner, N. and van der Laan, M. (2009). Biogenesis of precipitated with 20% trichloroacetic acid for 30 minutes on ice. mitochondrial membrane proteins. Curr. Opin Cell Biol. 21, 484-493. The supernatants were then spun at 14,000 ϫ g for 10 minutes at Bolender, N., Sickmann, A., Wagner, R., Meisinger, C. and Pfanner, N. (2008). 4°C. These pellets were then resuspended in Thorner buffer, Multiple pathways for sorting mitochondrial precursor proteins. EMBO Rep. 9, 42-49. Chen, B., Dodge, M. E., Tang, W., Lu, J., Ma, Z., Fan, C. W., Wei, S., Hao, W., Kilgore, resolved by 17.5% SDS-PAGE, and visualized by autoradiography. J., Williams, N. S. et al. (2009). Small molecule-mediated disruption of Wnt- dependent signaling in tissue regeneration and cancer. Nat. Chem. Biol. 5, 100-107. Whole-mount in situ hybridization Crosnier, C., Vargesson, N., Gschmeissner, S., Ariza-McNaughton, L., Morrison, A. and Lewis, J. (2005). Delta-Notch signalling controls commitment to a secretory fate Whole-mount in situ hybridization was performed, as described in the zebrafish intestine. Development 132, 1093-1104. previously (Alexander et al., 1998), using the wnt2bb (Ober et al., Curado, S., Anderson, R. M., Jungblut, B., Mumm, J., Schroeter, E. and Stainier, D. 2006) and tomm22 probes. To generate the tomm22 in situ probe, Y. (2007). Conditional targeted cell ablation in zebrafish: a new tool for regeneration the last exon and 3Ј-UTR of tomm22 were cloned into the pGEM- studies. Dev. Dyn. 236, 1025-1035. Curado, S., Stainier, D. Y. and Anderson, R. M. (2008). Nitroreductase-mediated T Easy vector (Promega). cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies. Nat. Protoc. 3, Chemical treatments 948-954. gz12 Curran, S. P., Leverich, E. P., Koehler, C. M. and Larsen, P. L. (2004). Defective tomm22 MO-injected Tg(fabp10:RFP) larvae exhibiting a mitochondrial protein translocation precludes normal Caenorhabditis elegans degenerating liver at 4 dpf were treated with 5 M of the Wnt–- development. J. Biol. Chem. 279, 54655-54662. catenin signaling pathway inhibitor IWR-1 (a gift from Lawrence Daum, G., Gasser, S. M. and Schatz, G. (1982). Import of proteins into mitochondria. Energy-dependent, two-step processing of the intermembrane space enzyme DMM Lum, University of Texas Southwestern Medical Center) (Chen et cytochrome b2 by isolated yeast mitochondria. J. Biol. Chem. 257, 13075-13080. al., 2009) in 0.05% dimethyl sulfoxide (DMSO), or with 0.05% Deas, T. S., Bennett, C. J., Jones, S. A., Tilgner, M., Ren, P., Behr, M. J., Stein, D. A., DMSO (carrier control) in embryo medium, at 28°C. A 1 mM stock Iversen, P. L., Kramer, L. D., Bernard, K. A. et al. (2007). In vitro resistance selection of the Fgf inhibitor SU5402 (Calbiochem) was prepared in 100% and in vivo efficacy of morpholino oligomers against West Nile virus. Antimicrob. Agents Chemother. 51, 2470-2482. DMSO and diluted to 1 and 3 M with egg water; the 0.1% DMSO Endo, T., Yamamoto, H. and Esaki, M. (2003). Functional cooperation and separation solution in egg water was used as a control. of translocators in protein import into mitochondria, the double-membrane bounded organelles. J. Cell Sci. 116, 3259-3267. ACKNOWLEDGEMENTS Farooq, M., Sulochana, K. N., Pan, X., To, J., Sheng, D., Gong, Z. and Ge, R. (2008). We thank Nikolaus Pfanner, Duc Dong, Michel Bagnat, Cagla Eroglu and members Histone deacetylase 3 (hdac3) is specifically required for liver development in of the Stainier lab for discussions; Markus Bussen, Isla Cheung and Stephanie zebrafish. Dev. Biol. 317, 336-353. Woo for comments on the manuscript; Bruce Draper, Natasha Zvenigorodsky, Fausto, N., Laird, A. D. and Webber, E. M. (1995). Liver regeneration. 2. Role of Donghun Shin, Linda Setiawan, Koraboshka Brand, Chong Hyun Shin, as well as growth factors and cytokines in hepatic regeneration. FASEB J. 9, 1527-1536. Sandra Huling and the UCSF Liver Center, for experimental assistance; Ana Ayala Fausto, N., Campbell, J. S. and Riehle, K. J. (2006). Liver regeneration. Hepatology 43, and Milagritos Alva for expert help with the fish; Julian Lewis and Jan Rombout S45-S53. for antibodies; and Lawrence Lum and Jim Amatruda for the IWR-1 compound. Field, H. A., Ober, E. A., Roeser, T. and Stainier, D. Y. R. (2003). Formation of the P.C-H. thanks Jodi Nunnari for support. This work was supported in part by digestive system in zebrafish. I. Liver morphogenesis. Dev. Biol. 253, 279-290. postdoctoral fellowships from the CVRI (S.C.), the American Heart Association Geller, B. L. (2005). Antibacterial antisense. Curr. Opin. Mol. Ther. 7, 109-113. (E.A.O.) and the Human Frontier Science Program (H.V.), and grants from the NIH Gerloff, T., Stieger, B., Hagenbuch, B., Madon, J., Landmann, L., Roth, J., Hofmann, (DK60322) and the Packard Foundation to D.Y.R.S. Deposited in PMC for release Disease Models & Mechanisms A. F. and Meier, P. J. (1998). The sister of P-glycoprotein represents the canalicular after 12 months. bile salt export pump of mammalian liver. J. Biol. Chem. 273, 10046-10050. COMPETING INTERESTS Goessling, W., North, T. E., Lord, A. M., Ceol, C., Lee, S., Weidinger, G., Bourque, C., The authors declare no competing financial interests. Strijbosch, R., Haramis, A. P., Puder, M. et al. (2008). APC mutant zebrafish uncover a changing temporal requirement for wnt signaling in liver development. Dev. Biol. AUTHOR CONTRIBUTIONS 320, 161-174. S.C. designed and performed experiments, analyzed the data and wrote the Guo, Y., Cheong, N., Zhang, Z., De Rose, R., Deng, Y., Farber, S. A., Fernandes- manuscript. E.A.O. analyzed data and contributed reagents and to the writing of Alnemri, T. and Alnemri, E. S. (2004). Tim50, a component of the mitochondrial the manuscript. S.W. designed and performed experiments, analyzed data and translocator, regulates mitochondrial integrity and cell death. J. Biol. Chem. 279, contributed to the writing of the manuscript. P.C.-H. contributed with data 24813-24825. analysis. H.V. contributed with reagents. C.M.K. designed experiments and Hayashi, T., Mizuno, N. and Kondoh, H. (2008). Determinative roles of FGF and Wnt analyzed data. D.Y.R.S. designed experiments, analyzed the data, wrote the signals in iris-derived lens regeneration in newt eye. Dev. Growth Differ. 50, 279-287. manuscript and was the principal supervisor. All authors read and commented on Heasman, J. (2002). Morpholino oligos: making sense of antisense? Dev. Biol. 243, 209- the manuscript. 214. Her, G. M., Chiang, C. C., Chen, W. Y. and Wu, J. L. (2003). 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