Early Developmental Pathology Due to Cytochrome cOxidase Deficiency Is Revealed by a New Zebrafish Model*□S

Received for publication, April 27, 2007, and in revised form, August 6, 2007 Published, JBC Papers in Press, August 30, 2007, DOI 10.1074/jbc.M703528200

Katrina N. Baden‡, James Murray§, Roderick A. Capaldi‡§, and Karen Guillemin‡1

From the ‡Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229 and §MitoSciences Inc.,Eugene, Oregon 97403-2095

Deficiency of cytochrome c oxidase (COX) is associated withsignificant pathology in humans. However, the consequencesfor organogenesis and early development are not well under-stood. We have investigated these issues using a zebrafishmodel. COX deficiency was induced using morpholinos toreduce expression of CoxVa, a structural subunit, and Surf1, anassembly factor, both of which impaired COX assembly. Reduc-tion of COX activity to 50% resulted in developmental defects inendodermal tissue, cardiac function, and swimming behavior.Cellular investigations revealed different underlying mecha-nisms. Apoptosis was dramatically increased in the hindbrainand neural tube, and secondary motor neurons were absent orabnormal, explaining the motility defect. In contrast, the heartlacked apoptotic cells but showed increasingly poor perform-ance over time, consistent with energy deficiency. The zebrafishmodel has revealed tissue-specific responses to COX deficiencyand holds promise for discovery of new therapies to treat mito-chondrial diseases in humans.

Cytochrome c oxidase (COX)2 is the terminal enzyme in themitochondrial respiratory chain. Embedded in the inner mito-chondrial membrane, COX couples the vectoral movement ofprotons across the inner membrane to the transfer of electronsfrom reduced cytochrome c to molecular oxygen. MammalianCOX consists of 13 subunits, 10 of which (COXIV–COXVIII)are encoded by nuclear DNA. The other three subunits (COXI,COXII, and COXIII) are encoded by the mitochondrial DNA(mtDNA) and constitute the hydrophobic/catalytic core of theenzyme. Assembly of COX is a complicated process involvingmultiple assembly factors and chaperones, including COX10–11, COX15–20, COX23, SCO1, SCO2, and SURF1 (1). Defi-

ciency in COX activity is associated with significant pathologyusually affecting highlymetabolic tissues, including brain,mus-cle, and eyes (2, 3). For example, COX deficiency is commonlyassociatedwith Leigh syndrome, an early-onset disorder result-ing in progressive central nervous system degeneration andearly lethality (4, 5). The majority of cases of COX-deficientLeigh syndrome are a result of mutations in SURF1, whichencodes a COX assembly factor (6, 7). Mutations in SURF1impair the incorporation of subunit II, resulting in a buildup ofan early COX assembly intermediate, decrease in other COXsubunits, and a decrease in COX activity (8–11). COX defi-ciencymay also be complicated by cardiac pathology, includinghypertrophic and dilated cardiomyopathies (12, 13), and con-duction defects (14).COX deficiency is detrimental during the prenatal period as

well. The incidence of this disorder has prompted the applica-tion of COX activity assays to prenatal samples obtained bychorionic villus biopsy and amniocentesis in specific cases (15,16). Prenatal diagnostics of this type have preparatory value forparents and physicians andmay also be used to explain previouspregnancy losses (17). However, our incomplete understandingof the metabolic requirements of different tissues and organsduring development diminishes the predictive value of theseassays.Initial studies using specific inhibitors of COX suggest that

there is a large reserve of COX activity. The biochemicalthreshold of COX inhibition that can be tolerated beforemitochondrial respiration is impaired was observed to be60–86% in isolated rat mitochondria (18–22). Studies inhuman cell lines using a specific COX inhibitor and mtDNAlesions revealed a range of thresholds from 21% to 82%depending upon cell type (23, 24). Clinically, patient sampleswith COX deficiencies between 50 and 85% are associatedwith severe pathologies (25–29), implying that the biochem-ical threshold of COX impairment is exceeded. However,questions as to how these levels of COX deficiency affect arapidly developing organism, tissue and organ sensitivity,and cellular responses remain unanswered. A vertebrate ani-mal model of COX deficiency is therefore desirable.Recently mouse has been used as a model system for COX

deficiency. Agostino et al. (30) generated a mouse null in Surf1.The Surf1 knock-outmouse exhibited defectiveCOXactivity intissue samples, muscle weakness, motor deficits, and early-post-natal lethality. However, there was �90% post-implanta-tion embryonic lethality, and the 10% of mice that survivedembryogenesis did not display any obvious neurological symp-toms. The relative inaccessibility of in uteromouse embryonic

* This work was supported by National Institutes of Health (NIH) Grants R21DK067065-01 (to K. G.) and RO1 DK075549-01 (to K. G.) and The Eugeneand Clarissa Evonuk Memorial Graduate Fellowship in Environmental orStress Physiology (to K. B.). NIH Grant HD22486 provided support for theOregon Zebrafish Facility. The costs of publication of this article weredefrayed in part by the payment of page charges. This article must there-fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental supplemental Fig. S1 and Movies S1 and S2.

1 To whom correspondence should be addressed: University of Oregon, 1370Franklin Blvd., Eugene, OR 97403-2095. Tel.: 541-346-5360; Fax: 541-346-5891; E-mail: guillemin@molbio.uoregon.edu.

2 The abbreviations used are: COX, cytochrome c oxidase; MO, morpholino;hpf, hours postfertilization; dpf, days postfertilization; rtPCR, reverse tran-scription-PCR; qrtPCR, quantitative rtPCR; mtDNA, mitochondrial DNA;PBS, phosphate-buffered saline; GFP, green fluorescent protein.

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development precludes simultaneous biochemical analysis andthorough investigation of organ development and functionduring the prenatal period, when the vast majority of the Surf1knock-out mice died. Furthermore, the cost and technicalrequirements of generating transgenic mouse mutants impedethe creation of allelic series of mutants with different levels ofreduction of COX components, which are needed to pinpointthe amount that best recapitulates humandisorders. Therefore,we chose to dissect the mechanisms of COX deficiency associ-ated with early developmental pathology and lethality in azebrafish model.Zebrafish develop ex utero protected from the outside

environment by a transparent chorion. Embryonic and larvalzebrafish tissue is also transparent, a trait that has madezebrafish a powerful tool for studying development andphysiology in real-time (31, 32). Furthermore, comparedwith mouse, zebrafish is readily amenable to the analysis ofdose-dependent loss of function phenotypes using antisensemorpholino (MO) oligonucleotides. MOs are gene-specificand block expression by binding RNA and sterically inhibit-ing either translation (33) or splicing (34). They can betitrated to inhibit protein expression to various degrees,resulting in a range of phenotypes from wild type to com-plete loss of function. The full spectrum of genetic mutationsthat precipitate COX deficiency is unknown. It is likely thatcertain mutations, or subsequent protein deficits, have notbeen identified, because they are necessarily lethal prena-tally. This may explain why no patients have been described todate with a mutation in a nuclear DNA-encoded structuralcomponent of COX (35). TitratedMO knockdown is thereforea promising means by which the requirement for certain geneproducts could be identified.In the current work we demonstrate that MOs are an effec-

tive means of inducing COX deficiency in zebrafish. We showthat MOs designed to reduce expression of Surf1, an assemblyfactor, and CoxVa, a structural component of COX, effectivelyimpair COX activity in developing zebrafish. We describe det-rimental effects of COX deficiency on cell biology, organ devel-opment, and organism physiology. These include increasedactivation of programmed cell death, defective secondarymotor neuron development, impaired endodermal organdevelopment, and cardiovascular dysfunction. The progressivedevelopmental pathologies culminate in cardiac failure andearly lethality. These data provide insights into the mechanismof COXdeficiency associatedwith embryonic loss and develop-mental pathology in humans. The utility of this zebrafishmodelis enhanced by its potential application in forward geneticscreens and high throughput small molecule screens to dis-cover modifier genes of COX activity and potential treatmentsof COX deficiency, respectively.

EXPERIMENTAL PROCEDURES

Zebrafish Maintenance and Care

Zebrafish (Danio rerio) embryos were collected from naturalcrosses of genotype ABC or Tg(gata2:GFP) adults at the Uni-versity of Oregon Zebrafish Facility. Embryos were reared at28.5 °C on a 14-h light/10-h dark cycle according to standard

protocols (36). Embryos were staged by hours postfertilization(hpf) and days postfertilization (dpf) according to Kimmel et al.(32). All zebrafish experiments were performed according toprotocols approved by the Institutional Animal Care and UseCommittee.

Morpholino and RNA Injections

Translation and splice-blockingMOswere designed byGeneTools, LLC. MOs were solubilized in sterile water then dilutedto working concentrations and injected as in Nasevicius et al.(33). A 1% phenol red solution was added to working concen-trations for visualization of injection volume. A translation-blocking MO, which binds at the ATG start, was designedagainst cox5aa (5�-ACAGTCGAAGGGCGGCTCGGAA-CAT-3�). The cox5ab-MO (5�-AGCTGGAGAACACAGC-GAACACACA-3�), which binds at the second exon spliceacceptor site, and surf1-MO (5�-CGGAAGGCACAGAGACT-CACTCTAC-3�), which binds at the fourth exon splice donorsite, were designed to block appropriate splicing of targetmRNA. These MOs had a 3�-fluorescein modification, and flu-orescence was used to select fish that had homogeneouslyincorporated theMOs. A splice-blocking galT-MO (5�-AAAT-CATTATGCACTCACCTGATGG-3�), which binds the sec-ond exon splice donor site of �1,3-galctosyltransferase, a non-mitochondrial enzyme, was used to control for nonspecificeffects of injection. p53-MO (5�-GCGCCATTGCTTTGCAA-GAATTG-3�), a translation blocker, was used to inhibit p53-dependent apoptosis (37). emx3-MO (5�-GACGTGTCT-GTCTCTTACCTGCGTC-3�), which binds the second exonsplice acceptor site of the empty spiracles homeobox 3 gene, wasused as a positive control for the effects of p53-MO.

mRNA Synthesis

cox5aamRNA with silent mutations at the MO binding siteand surf1mRNAwere transcribed out of pcDNA3 using the T7mMessagemMachine kit (Ambion) according tomanufacturerinstructions. Rescue experiments were performed by co-inject-ing cox5aa and surf1 mRNA with cox5aa-MO and surf1-MO,respectively. The amount of mRNA injected was titrated todetermine the effective dose.

PCR

rtPCR—3-dpf zebrafish samples were homogenized inTRIzol reagent (Invitrogen) using a Kontes pellet pestle. RNAwas isolated by chloroform extraction, isopropanol precipita-tion, andwashed in 75% ethanol. Contaminating genomicDNAwas removed with the DNA-free kit (Ambion). RNA wasreverse transcribed to cDNA using the Superscript III FirstStrand Synthesis Kit (Invitrogen) with oligo(dT). The followingprimers were used for amplification of a surf1 fragment span-ning theMO binding site: forward, 5�-GTCAACAATCAACT-TCCACTGTCG-3�; reverse, 5�-TTTGCTCCACTTTCAC-CACTGG-3�. rtPCR was carried out in an MJ Research PTC-225 Peltier Thermal Cycler. The cycling parameters were 95 °Cfor 5 min then 35 cycles of 94 °C for 1 min, 56 °C for 1 min, and72 °C for 1 min, and finally 72 °C for 10 min, and then held at10 °C.

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qrtPCR—cDNA was synthesized from extracted RNA asdescribed above. Genomic DNAwas extracted from samples of15 zebrafish at 3 dpf using a DNeasy Tissue Kit (Qiagen). Thefollowing primers were used in quantitative real-time rtPCRreactions: cox1 forward, 5�-ACTTAGCCAACCAGGAGCAC-3�; cox1 reverse, 5�-GGGTGGAAGAAGTCAGAAGC-3� (38);�-actin forward, 5�-TTCTGGTCGGTACTACTGGTATT-GTG-3�; and �-actin reverse, 5�-ATCTTCATCAGGTAGTC-TGTCAGGT-3� (38). Reactions were carried out in 25-�l vol-umes with 1� SYBER Green PCR Master Mix (MolecularProbes), 7.5 pmol of both forward and reverse primers, and 200ng of DNA. All samples were run in triplicate. qrtPCR was per-formed in 96-well plate format in an ABI Prism 7900HTsequence detection system (Applied Biosystems). Cyclingparameters were 50 °C for 2 min, 95 °C for 10 min, and then 40cycles of 95 °C for 15 s and 60 °C for 1 min. The run finishedwith 95 °C for 15 s followed by a dissociation step of 60 °C for15 s.

Microscopy

Images of live zebrafish were obtained with a digital cameraon a Zeiss Axioplan. Fluorescence images of live stained andfixed fish were captured on a Nikon Eclipse TE2000-U usingepifluorescence or confocal lasers. Adobe Photoshop was usedto adjust image brightness and contrast.

Immunohistochemistry

Zebrafish embryos were manually dechorionated and eutha-nized by an overdose of tricaine methane sulfonate (36).Embryoswere fixed in 4% paraformaldehyde BT fix (36). 24-hpfand 31-hpf fixed embryos were permeabilized in 0.5% TritonX-100 in phosphate-buffered saline (PBS). 48-hpf fixedembryos were permeabilized in 100% acetone at �20 °C for 25min. Permeabilized embryos were washed with 0.2% Tween 20,1% bovine serum albumin, and 1% Me2SO in PBS and blockedin wash solution with 2% normal goat serum. Embryos wereincubated with primary antibodies overnight at 4 °C. For pri-mary motor neuron detection 24-hpf embryos were incubatedwith zn1 monoclonal antibody (1:200) (39) and znp1 mono-clonal antibody (1:1000) (39), which were detected with goatanti-mouse Alexa 488 IgG1 and IgG2a (Molecular Probes),respectively. Secondary motor neurons were detected inTg(gata2:GFP) transgenic zebrafish by incubation with rabbitpolyclonal anti-GFP (1:200,Molecular Probes) followed by goatanti-rabbit Alexa 488 (Molecular Probes). Initiation of apopto-sis was detected by incubation with rabbit monoclonal anti-active caspase-3 (1:200, BD Pharmingen) (40) followed by goatanti-rabbit Alexa 594 (Molecular Probes). 4�,6-Diamidino-2-phenylindole nuclear stain (1:1000) was added to the second tolast wash.

Acridine Orange Staining

The vital dye acridine orange was used to identify apoptoticcells (41). 24-hpf, 31-hpf, 48-hpf, and 3-dpf live zebrafish weresoaked in 0.5 �g/ml acridine orange for 15min and washed fivetimes in embryomedium (36) at room temperature in the dark.

Western Blotting

20 zebrafish embryos at 3 dpf were homogenized in 100 �l of50 mM Tris buffer, pH 7.5, with a protease inhibitor mixture(Sigma-Aldrich) using a Kontes pellet pestle. Samples were fur-ther disrupted by brief sonication with a Branson Sonifier 150.SDSwas added to a final concentration of 0.5%. Insolublemate-rial was removed by centrifugation at 25,000 � g, and then theprotein concentration was determined by BCA assay accordingto manufacturer instructions (Pierce). Samples were electro-phoresed on a 10–20% acrylamide gel using the Laemmli buffersystem (42). Proteins were transferred from one-dimensionalgels to polyvinylidene difluoride membranes (0.45-�m poresize) as described previously (43).Membraneswere blocked in a5%milk/PBS solution overnight. Blots were probedwithmono-clonal antibodies against COXI and COXVa (MitoSciencesInc.) and a porin Ab-2 antibody (Calbiochem), all at a finalconcentration of 1 �g/ml in 1%milk/PBS solution. After wash-ing three times for 5 min with 0.05% Tween 20 in PBS, goatanti-mouse secondary antibody conjugated to horseradish per-oxidase (Jackson Immunolabs) was applied. Blots were visual-ized using the ECLPlusWestern blottingDetection System (GEHealthcare).

COX Activity

The substrate of the reaction, reduced ferrocytochrome c,was generated by dissolving 250mgof bovine heart cytochromec (Sigma) in 1 ml of 50 mM KH2PO4, pH 7.2, and reduced byaddition of L-(�)-ascorbic acid (MCB Reagents). Reducedbovine heart cytochrome c was purified at 4 °C over 20 ml ofSephadex G-25 (Amersham Biosciences) in a column equili-brated with 50mMKH2PO4, pH 7.2, and collected in 1-ml frac-tions before concentration measurement at 550 nm in a Beck-manDU7 (e� 19.1mM�1 cm�1). Zebrafish were homogenizedusing a Kontes pellet pestle and suspended to 5mg/ml in 50mMKH2PO4, pH7.2. Proteinswere detergent-extracted by adding 2mM dodecyl-�-D-maltoside and placed on ice for 20 min, andthen insoluble material was removed by centrifugation at25,000 � g for 20 min. This zebrafish detergent lysate wasdiluted to the desired amount for each well in 100 ml of 20 mMKH2PO4, pH 7.2, 0.3 mM dodecyl-�-D-maltoside. An equal vol-ume of 20mMKH2PO4, pH 7.2, 0.3mM dodecyl-�-D-maltoside,60 mM ferrocytochrome c was added to each well. The oxida-tion of the added ferrocytochrome cwas immediatelymeasuredat 30 °C using a Molecular Dynamics Spectramax 384 micro-plate reader (PerkinElmer Life Sciences) at 550 nm. The oxida-tion of ferrocytochrome c was followed for 105 min. However,the activity rates were calculated from linear slope of the initialrates at time points �11 min.

RESULTS

Zebrafish Have Two Copies of cox5a and One Copy of surf1Genes—The sequence identified as NM_001024403 was recov-ered from an initial BLAST of the zebrafish genome, having74% amino acid sequence identity to human COXVa (supple-mental Fig. S1A). Knockdown of zebrafish cox5a with a MOdesigned against this sequence successfully impaired COXactivity and development as described below. However, resid-ual CoxVa protein was detected by Western blot. Therefore,

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another BLAST search of the zebrafish genome was performedto look for a second cox5a gene. Due to an ancestral genomeduplication, zebrafish have duplicate co-orthologs for a mini-mum of 20% of human genes (44). We identified a secondsequence, XM_695484, as being 65% identical to humanCoxVaand 76% identical to the other zebrafish gene at the amino acidlevel (supplemental Fig. S1A). NM_001024403 was namedcox5aa and a translation blocking MO, cox5aa-MO, wasdesigned against the ATG start site of this sequence.XM_695484 was named cox5ab, and a splice-blocking MO,cox5ab-MO, was designed against the second exon spliceacceptor site. cox5aa and cox5ab should encode functionallyequivalent proteins. Here we refer to both proteins as CoxVa,because they are not distinguished by the antibody to humanCOXVa.In a BLAST search of the zebrafish genome for surf1 one

sequence, XM_678665, was identified with significant similar-ity to human SURF1. In a protein alignment, zebrafish Surf1 has60% amino acid sequence identity to the homologous humansequence (supplemental Fig. S1B). A search for a duplicate generecovered no other sequences with significant similarity. Asplice-blockingMO, surf1-MO,was designed against the fourthexon splice donor site of this gene.Reduction of Either CoxVa or Surf1 Impairs COX Activity—

Assembly of the COX holoenzyme requires the proper struc-tural subunits and necessary assembly factors. Therefore, MOreduction of either CoxVa or Surf1 was expected to alter theamount of normally assembled and functional COX. To deter-mine the effects of cox5aa-MO, cox5ab-MO, and surf1-MO ontotal zebrafish COX activity, we measured the initial rate ofcytochrome c oxidation by extracted zebrafish proteins fromuninjected and MO injected samples of 20–30 fish at 3 dpf.Zebrafish embryos were injected with 20 ng of cox5aa-MO,cox5ab-MO, or surf1-MO. In titration experiments this doseresulted in a reproducible phenotype (described below) thatwas not lethal at 5 dpf in the majority of injected zebrafish.Another control group received 20 ng of galT-MO, whichblocks splicing of �1,3-galactosyltransferase, a non-mito-chondrial gene.3 The COX activity in each MO-injectedpopulation was measured relative to an uninjected control inat least two separate experiments. For equivalent amounts ofprotein, COX activity in 3-dpf cox5aa-MO, cox5ab-MO, andsurf1-MO samples was approximately half that of uninjectedand galT-MO controls (as shown in a representative exper-iment, Fig. 1, A and B).Morpholino Reduction of Zebrafish cox5aa, cox5ab, and surf1

Results in Reduced COX Subunits—SURF1-deficient humanpatient cells exhibit decreased levels of COX protein subunits(9, 10), presumably due to impaired COX assembly and subse-quent proteolysis of subunits. We proposed that deficient pro-duction of the structural subunit CoxVawould similarly impairCOX assembly in zebrafish. Using monoclonal antibodies tothe human COX proteins that we showed cross-reacted withthe zebrafish proteins of the predicted size (data not shown), weassessed the levels of CoxVa and CoxI in cox5aa-MO, cox5ab-

MO, surf1-MO, and control samples inWestern blots. The lev-els of CoxVa andCoxIwere normalized to porin, an outermito-chondrial membrane protein, the level of which would not beexpected to be altered. Proteins were extracted from samples of�20 zebrafish at 3 dpf. As expected, the MO target protein,CoxVa, was decreased in cox5aa-MO and cox5ab-MO fish rel-ative to porin comparedwith uninjected and galT-MOcontrols(Fig. 1C). A concomitant decrease inCoxI, relative to porin, wasalso noted.Because no antibody was available to Surf1 in zebrafish to

confirm MO inhibition of protein expression, we designedsurf1-MO to inhibit splicing of the transcript. cDNA was pre-pared from RNA extracted from 3-dpf uninjected and surf1-MO-injected embryos and used in rtPCR with primers to thethird and fifth exons, which flank theMO binding site. A largerfragmentwas PCRamplified in the surf1-MOsample indicatingfailure to splice out the fourth intron (Fig. 1D). Western blot ofuninjected and surf1-MO zebrafish revealed a decrease in CoxIrelative to porin in the surf1-MO fish (Fig. 1E). The decrease inmtDNA-encoded CoxI in cox5aa-MO, cox5ab-MO, and3 J. Bates and K. Guillemin, unpublished data.

FIGURE 1. Morpholino knockdown of cox5aa, cox5ab, and surf1 impairsCOX activity and results in reduced COX subunits. A and B, the rate ofoxidation of ferrocytochrome c by extracted zebrafish protein from samplesof 20 uninjected, galT-MO, cox5aa-MO, cox5ab-MO, and surf1-MO zebrafish at3 dpf. COX activity was normalized to the uninjected control. C and E,zebrafish lysates were electrophoresed on 10 –20% acrylamide gels underdenaturing conditions. Membranes were blotted with monoclonal antibod-ies to CoxI, CoxVa, and porin. D, cDNA from 3-dpf uninjected and surf1-MOembryos was PCR-amplified using primers to the third and fifth exons of surf1.The larger amplicon in the surf1-MO sample reflects failure to splice out thefourth intron. F, real-time quantitative rtPCR amplification of cox1 and �-actinon cDNA synthesized from RNA extracted from 3-dpf uninjected, galT-MO,cox5aa-MO, cox5ab-MO, and surf1-MO zebrafish. The ratio of cox1 to �-actintranscripts in the uninjected sample was set to one, and the other samples aredescribed relative to the uninjected control.

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surf1-MO zebrafish indicates that COX assembly is impairedby reduced production of either a structural subunit or anassembly factor and that altered COX assembly results in sec-ondary loss of other mtDNA encoded subunits in zebrafish.mtDNA Transcript Levels Are Elevated in surf1-MO but Not

cox5aa-MO or cox5ab-MO Zebrafish—The decrease in CoxIdetected in cox5aa-MO, cox5ab-MO, and surf1-MO zebrafishcould be due to proteolysis of unincorporated subunits and/ordecreased mitochondrial biogenesis. To determine whethercox1 transcript abundance was altered by MO reduction ofCoxVa or Surf1, quantitative rtPCR (qrtPCR) was performedcomparing mtDNA encoded cox1 to nuclear DNA encoded�-actin on cDNA synthesized from RNA extracted from 10 to15MO injected and control zebrafish at 3 dpf. The ratio of cox1to �-actin transcripts was set to 1 in the uninjected samples.Comparedwith the uninjected control, the ratio of cox1:�-actintranscripts ranged from0.9 to 1.2 in galT-MOcontrols over fiveexperiments, 0.8 to 1.1 in cox5aa-MO fish over four experi-ments, and 0.9 to 1.1 in cox5ab-MO fish over five experiments(as shown in a representative experiment (Fig. 1F)). These datashow that transcript levels from mtDNA in fish injected witheither cox5aa-MO or cox5ab-MO were unaffected relative tocontrol samples. In contrast, the cox1:�-actin transcript ratio insurf1-MO injected fish ranged from 1.1 to 1.8 times controllevels over six experiments (as shown in a representative exper-iment, Fig. 1F), implying activation of compensatory mecha-nismswhen Surf1, but notCoxVa, is depleted. A similar up-reg-ulation of cox1 transcripts has been observed in COX-deficientpatient fibroblasts with SURF1mutations (9, 11).

The difference in transcript levels described above couldreflect changes in the number of functional mitochondria. Toquantify this, the cox1:�-actin ratio was measured by perform-ing qrtPCR on extracted genomic DNA from control and MOinjected 3-dpf zebrafish. Although much variability wasobserved between genomic extractions, no significant differ-ence was detected by one-way analysis of variance betweenuninjected andMO samples (data not shown). Therefore regu-lation of subunit levels occurs at the protein level in all COX-deficient samples, and in addition, compensatory transcriptregulation is observed in surf1-MO zebrafish.COX Deficiency Results in Abnormal Organ Development

and Physiology—The phenotypic effects of COX deficiency ondevelopment was assessed at 5 dpf, by which time wild-typezebrafish have a functional heart (45, 46), nervous system (47,48), and gut (49). The cox5aa-MO, cox5ab-MO, and surf1-MOfish all developed the same abnormal phenotypic traits. Theseincluded a shortened rostral-caudal body axis, no or uninflatedswim bladder, no or delayed gut development, edematous andunabsorbed yolk sac at 5 dpf, microphthalmia (small eyes), noor diminished jaw tissue, and abnormal head shape (Fig. 2, AandB). Larvae from all three COX-deficient populations exhib-ited no or reduced swimming spontaneously and in response totouch (Movie S1, a and b). Cardiovascular pathology consistedof significant pericardial edema (Fig. 2, C and D), bradycardia(reduced heart rate), ventricular asystole (failure to contract),and failure to develop into a loop (Movie S2, a and b). At 3 and4 dpf the ventricle was observed to contract in series with theatrium. The bradycardia and pericardial edema were progres-

sive, eventually leading to ventricular asystole at 5 dpf anddeathat�7dpf. The cardiac pathology is consistentwith the effects ofother non-respiratory chain mitochondrial perturbations inzebrafish (50, 51). Embryos injected with either buffer solutioncontaining phenol red or with 20 ng of galT-MO developedequivalently to uninjected embryos.Morpholino-resistant RNA Can Rescue the COX-deficient

Phenotype—To establish whether the described phenotype wasspecific to COX deficiency, MOs were co-injected with RNAdesigned to be resistant to the activity of the MO to test if theywould restore expression of the target gene product. For thetranslation blocking MO, cox5aa-MO, a cox5aa construct wasPCR-amplified with silent mutations in the MO binding site,from which mRNA could be translated and used in rescueexperiments. For the splice-blocking MO, surf1-MO, in vitrotranscribed target mRNA was co-injected to rescue thedescribed developmental phenotype. MOs were co-injectedwith a titration of the target mRNA for each experiment. Res-cue experiments for each MO were performed a minimum ofthree times.Phenotypes were scored at 5 dpf and qualitatively assessed as

being mild (pericardial edema alone and with mild head mal-formation and uninflated swim bladder), moderate (having thecomplete developmental and physiological COX-deficient phe-notype described in the previous section), and severe (exagger-

FIGURE 2. COX deficiency results in abnormal organ development andphysiology. A, normally developed 5-dpf uninjected zebrafish. B, 5-dpf COX-deficient zebrafish characterized by shortened rostral-caudal body axis, no oruninflated swim bladder, no or delayed gut development, microphthalmia,no or diminished jaw tissue, abnormal head shape, and yolk sac and pericar-dial edema. C, 5-dpf uninjected zebrafish heart. The ventricle is loopedbehind the atrium. D, 5-dpf COX-deficient zebrafish heart. The heart is notlooped and is surrounded by pericardial edema. sb, swim bladder; a, atrium;and v, ventricle.

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ation of the moderate phenotype with severe rostral-caudalbody axis truncation) (Fig. 3A). In a representative experiment,co-injection of 10 ng of cox5aa-MOwith 4 ng of cox5aamRNAdoubled the percentage of normally developed and mild phe-notype fish at 5 dpf compared with injection of 10 ng ofcox5aa-MOalone (Fig. 3B). Similarly, in another representativeexperiment, co-injection of 20 ng of surf1-MO with 0.5 ng ofsurf1mRNA increased the percentage of 5-dpf fish with a nor-mal phenotype by 4-fold compared with injection of 20 ng ofsurf1-MO alone (Fig. 3C).Efforts to rescue the cox5ab-MO phenotype with in vitro

transcribed cox5abmRNAwere unsuccessful (data not shown).This failure may have been due to an inability to isolate a clean

sample of cox5ab mRNA, com-pounded with the presence ofanother in-frame start codon up-stream of the presumed cox5abtranscriptional start. Although PCRamplification of cox5ab out ofzebrafish cDNA from this upstreamATG was not successful, we couldnot rule out this being a functionalstart codon.cox5aa-MO and surf1-MO Inter-

act Synergistically to Cause theCOX-deficient Phenotype—If theabnormal phenotype observed incox5aa-MO and surf1-MO fishwere due to COX deficiency, thenthere should be low doses ofcox5aa-MO and surf1-MO thatalone have little to no effect buttogether impair COX activity to adegree that can be read out in thephenotype. This hypothesis was

tested in two separate experiments. In a representative experi-ment, nearly all fish injected with either 1 ng of cox5aa-MO or3.75 ng of surf1-MO developed normally to 5 dpf. However,when 1 ng of cox5aa-MOwas co-injected with 3.75 ng of surf1-MO, 34% of fish exhibited the COX-deficient phenotype (Fig.4). If the phenotype described previously was the result of twodifferent metabolic or cellular impairments then a reduction inone pathway would have no effect on the end point of the otherpathway. Instead a combination of cox5aa-MO and surf1-MO,each below the minimum level needed for an individual effect,had a synergistic effect on phenotype. These data indicate that,although cox5aa-MO and surf1-MO have different gene-spe-cific actions, they target the same pathway.COX-deficient Zebrafish Exhibit Increased Apoptosis—Mito-

chondria play a pivotal role in progression through apoptoticpathways (52, 53). Therefore, COX-deficient mitochondrialpathology might alter the homeostatic levels of apoptosis thatnormally occur during development. We utilized fluorescentstains of apoptotic cells to visualize these effects. Live 24-hpf,31-hpf, 48-hpf, and 3-dpf zebrafish were stained with the vitaldye acridine orange, amarker of apoptotic cells (41).More apo-ptotic cells were observed in cox5aa-MO, cox5ab-MO, andsurf1-MO fish compared with uninjected and galT-MO con-trols at 24 hpf and most dramatically at 31 hpf, subsiding atsubsequent time points (Fig. 5, A–E, and data not shown).Apoptotic cells were stained throughout the head region, neu-ral tube, and trunk of developing COX-deficient fish but werenot detected in the heart.Because the acridine orange staining was most intense at 31

hpf, immunohistochemical staining of active caspase-3 wasperformed at this time point. The caspase-3 protease is acti-vated during early apoptosis and plays an essential role in theproteolytic cleavage of a number of proteins involved in apo-ptosis (54, 55). An increase in the amount of the active form ofcaspase-3 was observed in cox5aa-MO, cox5ab-MO, andsurf1-MO injected zebrafish compared with uninjected and

FIGURE 3. MO-resistant RNA can rescue the COX-deficient phenotype. cox5aa-MO and surf1-MO wereco-injected with in vitro transcribed target RNA resistant to the respective MO. Phenotypes were scored byseverity and compared with those resulting from MO injection alone. A, scale of COX-deficient phenotypeseverity. The mild phenotype includes pericardial edema alone and with uninflated swim bladder. The mod-erate phenotype includes all abnormalities described in Fig. 2. The severe phenotype is an exaggeration of themoderate phenotype with severe rostral-caudal body axis truncation. B, pie charts of phenotypes resultingfrom 10 ng of cox5aa-MO alone and 10 ng of cox5aa-MO with 4 ng of cox5aa mRNA with silent mutations in theMO binding site. C, pie charts of phenotypes resulting from 20 ng of surf1-MO alone and 20 ng of surf1-MO with0.5 ng of surf1 mRNA.

FIGURE 4. cox5aa-MO and surf1-MO interact synergistically to cause theCOX-deficient phenotype. The phenotypes of uninjected (n � 66) and fishinjected with 20 ng of galT-MO (n � 50), 20 ng of cox5aa-MO (n � 24), 20 ngof surf1-MO (n � 93), 1 ng of cox5aa-MO (n � 58), 3.75 ng of surf1-MO (n � 63),and 1 ng of cox5aa-MO plus 3.75 ng of surf1-MO (n � 70) were compared at 5dpf. Percentages of normally developed fish are listed above each sample bar.1 ng of cox5aa-MO and 3.75 ng of surf1-MO separately had little effect buttogether resulted in the COX-deficient phenotype.

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galT-MO controls, indicating progression through apoptoticpathways in COX-deficient zebrafish (Fig. 5, F–J). Activecaspase-3 staining was especially clustered in the head andhindbrain of COX-deficient fish.A p53-MO has been shown to block p53-dependent cell

death in zebrafish (37). Recently, Robu et al. (56) have pro-posed that an increase in p53-dependent cell death is anoff-target nonspecific effect of some MOs, and they showedthat it can be rescued by co-injection of a p53-MO of thesame sequence. Using a MO to empty spiracles homeobox 3(emx3-MO), which targets a transcription factor importantfor brain development (57) and has been previously shown toinduce such nonspecific cell death,4 we saw similar levels ofacridine orange staining as in surf1-MO-injected fish. Aspreviously observed by our colleagues, co-injection of thesame p53-MO with emx3-MO ameliorated the apoptosisdetectable with acridine orange staining in a dose-depend-ent manner. In contrast, when p53-MO was injected witheither 10 ng or 20 ng of surf1-MO, which induced high levels ofapoptosis, little to no amelioration of the apoptosis detected byacridine orange staining was observed. These data argue thatthe increased apoptosis observed in the COX-deficientzebrafish is not an off-target effect of MO injection.Motor Neuron Development Is Impaired in COX-deficient

Zebrafish—At 5 dpf cox5aa-MO-, cox5ab-MO-, and surf1-MO-injected fish had severely impaired motility, with no ordiminished swimming spontaneously and in response to touch(Movie S1, a and b). These motility defects, along with the

staining of apoptotic cells in theneural tube of COX-deficient fish at31 hpf (Fig. 5C), prompted us toexamine the effects of COX defi-ciency on motor neuron develop-ment. The axialmuscles of zebrafishare innervated by primary and sec-ondary motor neurons, which aredistinguishable by location, size,and axonal pathways (58). Primarymotor neurons develop earlier andare responsible for the spontaneouscontractions that can first beobserved at 17 hpf (59). At 26 hpfcox5aa-MO, cox5ab-MO, andsurf1-MO zebrafish displayed spon-taneous contractions that werecomparable to those seen in unin-jected and galT-MO controls(Movie S1, c and d). Furthermore,immunohistochemical assays usingprimary antibodies to zn-1 andznp-1, primary neuronal markers at24 hpf (39), confirmed normaldevelopment of primarymotor neu-ron structure in all three COX-defi-cient zebrafish populations (Fig. 6,A and B).

Axonal outgrowth of secondary motor neurons, which aremost similar to mammalian and avian motor neurons (60),occurs later than primary motor neurons (61). They are alsomore numerous, and some innervate more than one hemiseg-ment (58, 62). To assess the effects of COX deficiency on sec-ondary motor neuron development, cox5aa-MO, cox5ab-MO,and surf1-MO were injected into embryos of Tg(gata2:GFP), atransgenic line of zebrafish that express GFP in a subset of sec-ondary motor neurons and occasionally in descending inter-neurons and Rohon-Beard cells (63). To enhance the fluores-cent signal from secondary motor neurons, 48-hpf Tg(gata2:GFP) embryos were fixed and immunohistochemically stainedwith a primary antibody to GFP. Development of secondarymotor neurons in themajority of cox5aa-MO, cox5ab-MO, andsurf1-MO zebrafish was severely impaired (Fig. 6). Most axonsin the rostral two-thirds of the COX-deficient fish were absent(Fig. 6D). Other secondary motor neuron axons developed butwere stunted or irregularly spaced compared with uninjectedand galT-MO controls (Fig. 6E). Axonal disruptions were oftenaccompanied by a decrease in the number of secondary motorneuron cell bodies in the neural tube (Fig. 6F). The caudal one-third of the COX-deficient zebrafish generally displayed moresecondary motor neuron axons, and these were more likely tohave normal morphology than in the rostral portions. Thesedata may indicate an increased vulnerability of rostral second-ary motor neurons. Alternatively, as motor neurons developfrom rostral to caudal (64, 65), it may be that disruption ofsecondary motor neurons is a consequence of temporal bio-chemical derangements, possibly reflecting dilution anddecreased efficacy of the MOs.4 G. Aspock and M. Westerfield, unpublished data.

FIGURE 5. COX-deficient zebrafish exhibit increased apoptosis. Live 31-hpf zebrafish were stained withacridine orange, anesthetized with tricaine, and imaged with epifluorescence: uninjected (A), galT-MO (B),cox5aa-MO (C), cox5ab-MO (D), and surf1-MO (E). 31-hpf fixed zebrafish were stained with an active caspase-3antibody followed by goat-anti-rabbit Alex 594 (red) and 4�,6-diamidino-2-phenylindole (blue): uninjected (F),galT-MO (G), cox5aa-MO (H), cox5ab-MO (I), and surf1-MO (J). Scale bar: 300 �m for A–E and J; 240 �m for H andI. K, live 31-hpf zebrafish embryos were stained with acridine orange and visualized by epifluorescence. Stain-ing was scored from � to ���, where � represents minimal scattered stained cells and ��� indicatesstaining of apoptotic cells throughout the fish too numerous to count. Control fish were not injected with MO.One dot represents one fish, and the line signifies the group average.

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DISCUSSION

The effects of COX deficiency in humans are usually mani-fest in tissueswith highmetabolic activity.However, the clinicalpresentation of COX deficiency is variable in terms of the com-bination of tissues affected and severity of pathology. Onset ofpathology is generally early and has been detected in the prena-tal period. To date, an incomplete understanding of the geneticcauses of COX deficiency (66) and the variability in presenta-tion (35) have hindered our comprehension of pathophysiolog-ical mechanisms. Previous studies in mouse have yieldedimportant information, although the relative inaccessibility ofin utero development prevents a thorough dissection of theeffects of COX deficiency on early development. Additionally,the all-or-none phenotypes that result from deleting a genepreclude investigation of gene dosages required for develop-ment. Zebrafish offer an attractive alternative disease modelbecause they develop ex utero, are transparent in embryonicand larval stages, and rely on many of the same major organsand systems as humans (67). By titrating the injection of MOstargeting a COX structural subunit, CoxVa, and an assemblyfactor, Surf1, we have been able to reduce the level of COX

activity to a degree that impairs normal development but is notimmediately lethal. Capitalizing on the unique characteristicsof zebrafish, we have investigated the effects of COX deficiencyon organ development and function, revealing zebrafish as aninformative and powerful model of COX-deficient mitochon-drial disease.MOs designed against cox5aa, cox5ab, and surf1 reduced

COX activity in developing zebrafish to �50% of wild-type lev-els (Fig. 1, A and B). The accompanying decrease in CoxI pro-tein (Fig. 1, C and E) likely indicates altered enzyme assemblyand subsequent proteolysis of unused subunits, as has been sug-gested by work in SURF1-deficient human cell lines (10, 11).Levels of mtDNA-encoded cox1 transcripts in cox5aa-MO andcox5ab-MO fish were approximately equal to control samples,indicating little or no secondary effects of CoxVa reduction onmitochondrial biogenesis (Fig. 1D). However, the decrease inCoxI induced byMOreduction of Surf1was accompanied by anup-regulation of cox1 transcripts (Fig. 1E), which implies stim-ulation of a compensatory feedback mechanism in response tothe reduction of Surf1 but not CoxVa. Inadequate Surf1 mayitself instigate up-regulation of mtDNA transcripts. Alterna-tively, the loss of Surf1 likely stalls assembly of COX at a differ-ent point than loss of CoxVa, and the transcript level compen-sation may be secondary to the assembly effects. MammalianCOX assembly proceeds through a series of intermediates (68).The first is COXI alone followed by the addition ofCOXIV.Thenext phase involves incorporation of COXII, COXIII, and allnuclear encoded factors except subunits VIa and VIIa or VIIb,which are added in the last phase before dimerization.Work byStiburek et al. andWilliams et al. (69, 70) identified an assemblyintermediate composed of subunits I, IV, and Va, implying thatthe SURF1-mediated incorporation of subunit II occurs afterVa is added. According to this model, MO reduction ofzebrafish Surf1 would disrupt assembly at a later stage than lossof CoxVa, providing a possible temporal explanation for thediffering effects on mtDNA transcripts.MO reduction of either the CoxVa structural subunit or the

Surf1 assembly factor resulted in decreased COX activity to�50% of that in control fish (Fig. 1,A and B). Pooled samples oflive zebrafish with �57% COX impairment were not identifiedat 3 dpf. In contrast, studies performed on tissue samples andcell culture showed that COX activity could be impaired by asmuch as 82% in a human cell line (23) and 88% in isolated ratkidneymitochondria (22) without affecting cellular respiration.With these types of samples, however, threshold represents anarbitrary parameter of assaying mitochondrial throughputrather than a phenotypic determinant. An understanding of theeffects of cellular COXdeficiency on tissue development, organfunction, and organism survival requires a more complexmodel system. In our zebrafish model 50% impairment ofCOX activity was adequate for initial stages of zebrafishdevelopment, but it was insufficient for maintenance of cel-lular homeostasis, cardiac function, development of theperipheral nervous system, and was lethal by 7–9 dpf due tocardiac failure.Several controls were employed to demonstrate that these

effects were specific to COX deficiency and not secondary toinjection trauma orMO toxicity. First, three differentMOs tar-

FIGURE 6. Secondary motor neuron development is impaired in COX-de-ficient zebrafish, whereas primary motor neurons are spared. Immuno-histochemical staining of 24-hpf zebrafish with zn1 and znp1 antibodies fol-lowed by goat-anti-mouse Alexa 488 IgG1 and IgG2a revealed normalprimary motor neuron axons extending from the ventral spinal cord in unin-jected (A) and COX-deficient (B) zebrafish. 48-hpf Tg(gata2:GFP) zebrafishexpressing GFP in a subset of secondary motor neurons were fixed, immuno-histochemically stained with anti-GFP followed by goat-anti-rabbit Alexa 488,and visualized on a confocal microscope. C, uninjected zebrafish with nor-mally developed secondary motor neurons. COX-deficient zebrafish second-ary motor neuron phenotypes included lack of axons (D), stunted and irreg-ularly spaced axons with decreased lateral branches (E), and decreased cellbodies (F). Scale bar: 140 �m for A and B; 100 �m for C–F.

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geting COX factors, cox5aa-MO, cox5ab-MO, and surf1-MO,precipitated the same phenotype. Second, control fish injectedwith buffer containing phenol red or 20 ng of galT-MO, whichtargets a non-mitochondrial protein, developed the same asuninjected fish. Third, co-injections of MO with in vitro tran-scribed target mRNA rescued the COX-deficient phenotype,significantly increasing the percentage of normally developedfish at 5 dpf (Fig. 3). Fourth, the doses of cox5aa-MO andsurf1–MO could be titrated down to levels that individuallyhave no phenotypic effect but when injected together synergis-tically induce the COX-deficient phenotype (Fig. 4). Finally,co-injection of a target MOwith p53-MO has been reported toalleviate apoptosis associated with general MO off-targeteffects (56). We observed no significant reduction in the COX-deficient apoptosis in zebrafish co-injected with surf1-MO andp53-MO relative to surf1-MO alone (Fig. 5K), arguing againstthe phenotype being due to MO off-target effects.Our investigations into the progression of pathology that cul-

minates in the COX-deficient phenotype revealed a dramaticincrease in apoptosis, peaking at 31 hpf, in cox5aa-MO, cox5ab-MO, and surf1-MO samples (Fig. 5, A–E). Immunohistochem-ical staining of active caspase-3 showed that some, if not all, ofthis programmed cell death occurs through a caspase-depend-ent pathway (Fig. 5, F–J). Furthermore, co-injection of p53-MOwith surf1-MO failed to ameliorate the increase in apoptosis(Fig. 5K). Knockdown of p53 by MO technology may be insuf-ficient to block all p53-dependent cell death in a COX-deficientbackground. Alternatively, the observed increase in apoptosismay be p53-independent.Interestingly, apoptotic cells were not identified in the heart

of COX-deficient zebrafish, suggesting that the cardiac pathol-ogy and eventual failure is a functional pathology due to energyfailure, not an anatomic pathology secondary to cell loss. Thefact that apoptotic cells were not identified in the heart is alsoindicative of the different cellular responses to COXdeficiency,which may be a tissue-dependent phenomenon reflecting dif-ferent thresholds of tolerable COX impairment. Similarly, tis-sues mature at different rates and stages of organism develop-ment. The cellular energy requirements likely fluctuatedepending upon stage ofmaturation or differentiation andmaycontribute to whether cells with the same level of COX defi-ciency undergo apoptosis or survive but are functionallyimpaired. Also, cells in different tissues probably have differentsensitivities to stress and hence different propensities toundergo apoptosis in response to stress. Qualitatively, theCOX-deficient zebrafish hearts did not appear hypertrophic ordilated. However, it may be that the timeline over which heartfailure occurred was not long enough to result in sufficientphysiological feedback to precipitate identifiable changes incardiac wall or chamber size. Electrophysiological studieswould be required before attributing the observed ventricularasystole to heart block.In contrast to the heart, extensive apoptosis was observed in

the neural tube (Fig. 5C). This finding, alongwith the decreasedmotility phenotype at 5 dpf (Movie S1, a and b), prompted us toinvestigate the development of motor neurons in the COX-deficient background. Primarymotor neuron development wasunaltered by COX deficiency, as indicated by immunohisto-

chemical staining of primary motor neurons at 24 hpf (Fig. 6, AandB) and observations of normal spontaneous contractions at26 hpf in cox5aa-MO, cox5ab-MO, and surf1-MO zebrafish(Movie S1, c and d). Interestingly, development of secondarymotor neurons, which more closely resemble mammalianmotor neurons (60), was severely impaired in the COX-defi-cient zebrafish. Secondary motor neuron axons extend laterthan primary motor neurons (61) and some innervate morethan one hemisegment (58, 62). In most cases the majority ofsecondary motor neuron axons in the rostral two-thirds of theCOX-deficient fish were absent (Fig. 6D). In some cases short-ened and irregularly spaced axons were present (Fig. 6E). Sec-ondary motor neuron cell bodies were also reduced in number(Fig. 6F). The temporal disparity in development of primaryversus secondary motor neurons may account for the differenteffects of COX deficiency on these two populations of neurons.Thematernal contribution ofmitochondrial proteins and tran-scripts may be sufficient to allow normal development of pri-marymotor neurons but insufficient to compensate for theMOreduction ofCOXactivity during later secondarymotor neurondevelopment. Grunwald et al. (71) have shown that a ned-1mutation in zebrafish that results in central nervous system celldeath and prevents formation of secondarymotor neurons, buthas no effect on primary motor neuron development, is suffi-cient to prevent coordinated swimming activity. Additionally,Cheesman et al. (72) demonstrated that MO reduction ofNkx6.1, which also results in loss of secondary motor neuronsand spares primary motor neurons but does not increase celldeath, prevents swimming in response to touch. The lack ofsecondarymotor neurons and increased apoptosis in the COX-deficient zebrafish could therefore account for the impairedmotility at 5 dpf.Peripheral neuropathies have been described in a number of

human mitochondrial diseases with COX deficiency and orsequenced COX mutations (25, 73–76), including mitochon-drialmyopathy, encephalopathy, lactic acidosis, and stroke-likeepisodes (28), myoclonus epilepsy with ragged red fibers (76),and Leigh syndrome (26, 27, 77). In a study of 43 patients, Stick-ler et al. (76) concluded that peripheral neuropathy is a com-mon component of genetic mitochondrial disorders and islikely the result of dysfunctional cellular energy production.They propose that it is often unrecognized on clinical examina-tion because of confounding neuromuscular degeneration. Inhumans, the peripheral nervous system pathologies are gener-ally primary demyelination and axonal changes (26, 27, 29, 76,78). Based on our findings using a zebrafishmodel, we postulatethat COX deficiency during early development is particularlydetrimental to the peripheral nervous system, preventing eventhe development of motor neurons. COX deficiency is also det-rimental to cardiac function at this stage, which results in earlylethality. A similarly severe phenotype has been documented ina human infant with a mutation in SCO2, another COX assem-bly factor (79). This patient died of heart failure at 2 months ofage, and autopsy revealed moderate-to-severe motor neuronloss from the ventral horns of the spinal cord (77). Together,these data indicate that we should anticipate cardiac andperipheral nerve pathology following diagnosis of COX defi-ciency in prenatal and early post-natal patients. Similarly,mito-

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chondrial respiratory chain diseases should be included on a listof differential diagnoses for infants displaying signs of periph-eral neuropathy and cardiac pathology.Finally, zebrafish have been used in developmental screens

to identify small molecules that modify development (80).Here we have identified a COX-deficient developmentalphenotype that includes cellular, organ, and organismpathology. To date, there are only a limited number of palli-ative and supportive treatments of COX-deficient disease(81). The identification of a specific and titratable COX-deficient phenotype in zebrafish has promising future appli-cation in screens for small molecules that ameliorate thebiochemical and developmental pathologies.

Acknowledgments—We gratefully acknowledge the advice and reagentsprovided by Sarah Cheesman, Jennifer Bates, Gudrun Aspock, MonteWesterfield, and Judith Eisen; and Rose Gaudreau and the University ofOregon Zebrafish Facility for zebrafish husbandry.

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Katrina N. Baden, James Murray, Roderick A. Capaldi and Karen GuilleminRevealed by a New Zebrafish Model

Oxidase Deficiency IscEarly Developmental Pathology Due to Cytochrome

doi: 10.1074/jbc.M703528200 originally published online August 30, 20072007, 282:34839-34849.J. Biol. Chem. 

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