Brief Communication 1001

Zebrafish dracula encodes ferrochelatase and its mutationprovides a model for erythropoietic protoporphyriaSarah Childs*†, Brant M. Weinstein*†‡, Manzoor-Ali P.K. Mohideen*§, Susan Donohue*¶, Herbert Bonkovsky*¶ and Mark C. Fishman*

Exposure to light precipitates the symptoms of severalgenetic disorders that affect both skin and internalorgans. It is presumed that damage to non-cutaneousorgans is initiated indirectly by light, but this is difficultto study in mammals. Zebrafish have an essentiallytransparent periderm for the first days of development.In a previous large-scale genetic screen we isolated amutation, dracula (drc), which manifested as a light-dependent lysis of red blood cells [1]. We report herethat protoporphyrin IX accumulates in the mutantembryos, suggesting a deficiency in the activity offerrochelatase, the terminal enzyme in the pathway forheme biosynthesis. We find that homozygous drcm248

mutant embryos have a G→→T transversion at a splicedonor site in the ferrochelatase gene, creating apremature stop codon. The mutant phenotype, whichshows light-dependent hemolysis and liver disease, issimilar to that seen in humans with erythropoieticprotoporphyria, a disorder of ferrochelatase.

Address: *Cardiovascular Research Center, Massachusetts GeneralHospital East, Charlestown, Massachusetts 02129, USA.

Present addresses: ‡Laboratory of Molecular Genetics, National Instituteof Child Health and Development, Maryland 20892, USA. §Jake GittlenCancer Research Institute, The Pennsylvania State University College ofMedicine, Hershey, Pennsylvania 17033, USA. ¶The Center for Study ofDisorders of Iron and Porphyrin Metabolism and The Division ofDigestive Disease and Nutrition, University of Massachusetts MedicalSchool, Worcester, Massachusetts 01655, USA.

Correspondence: Mark C. FishmanE-mail: fishman@cvrc.mgh.harvard.edu

†These authors contributed equally to this work.

Received: 10 April 2000Revised: 16 June 2000Accepted: 3 July 2000

Published: 11 August 2000

Current Biology 2000, 10:1001–1004

0960-9822/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.

Results and discussionIn a large-scale mutant screen we identified several classesof recessive lethal mutations with reduced numbers of orno red blood cells [1]. One subset, including drc, exhibits afar more pronounced anemia when raised under standardambient light conditions than if raised in the dark. We iso-lated four alleles of drc: drcm87, drcm248 and drcm328 lackblood entirely four days post-fertilization (4 dpf) when raised

under normal light, and drcm159 has only a few circulatingcells at that time [1]. drc erythrocytes are strongly fluorescentwhen exposed to epifluorescent illumination using a rho-damine filter (peak illumination at 510–560 nm). Upon illu-mination, red cells immediately begin to lyse within theblood vessels, and completely disappear within 2 minutes(Figure 1a). Under standard microscopic white light illumi-nation, the lysis proceeds more slowly, and takes15–20 minutes to be complete. Rapid erythrocyte lysis hasother detrimental effects upon the embryo, manifest asbradycardia and diminished cardiac contractility. Thisappears to depend on the acuity of hemolysis, as embyros of

Figure 1

Blood and liver phenotypes of drc mutant zebrafish. (a) Time-lapseseries showing autofluorescent erythrocytes in the trunk of 1 dpfdrcm248 embryos visualized under fluorescent light with a rhodaminefilter (peak illumination at 510–560 nm). Circulation was arrested byuse of Tricaine. By 120 sec, all erythrocytes have lysed. (b) Side view of6 dpf drcm248 mutant and wild-type (WT) larvae under white light (toppanels) and fluorescent light (bottom panels). The reddish liver in themutants has a large number of red-brown inclusions that autofluoresce.(c) Cross-sections of 6 dpf drcm248 mutant and wild-type embryosunder fluorescent (left) and Nomarski (right) optics. Mutants showextensive autofluorescence in the liver, and pronephric ducts but not inother tissues. For sectioning, embryos were fixed in paraformaldehyde,dehydrated in an ethanol series and embedded in JB4 medium(Polysciences). Sections 5 µm thick were mounted without coverslipsand viewed with a rhodamine filter to detect autofluorescence. d,pronephric duct; g, gut; l, liver; nc, notochord; nt neural tube; s, somite.

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the same age exposed chronically to low light levels frombirth have a similar degree of anemia, but without abnormalheart rate or contractility.

The liver of drcm248 mutant embryos shows several hepaticabnormalities. It has a red hue and many inclusions arevisible under both white and fluorescent light (Figure 1b).The cells are pleiomorphic and show variable histologicalatypia (data not shown). The hepatic inclusions are notlimited to the region of vessels, but are scattered through-out the parenchyma (Figure 1c). In drcm248, these hepaticinclusions accumulate even in dark-raised embryos. Wealso observed fluorescent inclusions in the pronephricducts of 6 dpf drcm248 embryos. At 4 dpf, the zebrafishkidney becomes a major site of erythropoiesis, and thusthe accumulation of fluorescence there is likely to be dueto the presence of erythrocytes in the tissue.

The phenotype of drc mutants is suggestive of a group ofdisorders known as porphyrias in humans, which are causedby mutations in enzymes of the heme biosynthetic pathway.The sequential actions of eight enzymes generate heme, apathway initiated by condensation of glycine and succinylCoA into δ-aminolevulinate. Defects in the last fiveenzymes of the pathway are accompanied by accumulationof porphyrin intermediates. Illumination of these highlylight-sensitive compounds causes the production of singletoxygen and free radicals, leading, among other effects, tolipid peroxidation [2,3]. Patients show a constellation ofsymptoms which vary among porphyrias. They includecutaneous sensitivity to light, hemolysis of autofluorescent

red blood cells and hepatic pathology. A phenotype similarto that of drc has been observed in the zebrafish mutationyquem, which has a mutation in uroporphyrinogen decar-boxylase, an enzyme in this pathway [4]. drc is, by comple-mentation analysis, a different gene from yquem.

We examined porphyrin levels in drcm87 embryos byHPLC. One porphyrin in particular, protoporphyrin IX, ispresent at very high levels in drc mutant embryos (Figure 2)whereas it is nearly undetectable in a pool of wild-typeand heterozygous embryos. This suggested that the muta-tion in drc mutant embyros might lie in the ferrochelatase(fch) gene, which encodes an enzyme that converts proto-porphyrin IX into heme by catalyzing the transfer of ironto the heme moiety.

In humans, mutations in fch cause the disorder erythropoi-etic protoporphyria (EPP; OMIM entry 177000). Patientshave autofluorescent, light-sensitive red cells, and somedevelop severe liver disease. In humans, red cells arethought to be exposed to light during their transit throughthe skin, and it is presumed that the liver is damaged byuptake of toxic substances released by their lysis, as wellas from local synthesis of protoporphyrin [5]. Crystallinedeposits of protoporphyrin have been observed in thelivers of patients with EPP [6]. The inclusions we observein the liver of 6 day drc mutants may be similar to these,although they appear not to be biorefringent, and mayrepresent a stage before the formation of large crystals.

To determine whether fch is mutated in drc we first clonedthe wild-type zebrafish gene. The cDNA is 1405 bp longand encodes a putative 409-amino-acid protein (GenBankaccession number AF250368). The predicted proteinsequence is highly conserved through evolution, with a>80% identity to human and other vertebrate fer-rochelatases, 57% identity to the Drosophila gene, and 50%identity to the Saccharomyces cerevisiae gene.

In situ hybridization shows that expression of fch in thelateral plate mesoderm begins early in development, aroundthe 9–10 somite (S) stages. This is later than the earlyhematopoietic markers GATA2 and GATA1 [7], but con-temporaneous with other markers of differentiated erythro-cytes, such as aminolevulinate synthase (ALAS) [8]. At thistime, cells which will adopt a hematopoietic fate are inter-spersed in the lateral plate with cells which will becomevessel and pronephric duct. In fact, there may be a sharedcommon precursor cell for the first intra-embryonic bloodand angioblasts, a cell tentatively called the hemangioblast[9]. flk-1, fli-1 and scl are expressed by both hematopoieticand angioblast lineages [10,11]. At the 9–10S stage, fch isexpressed in the trunk (but not the head) of the zebrafishembryo in a pattern similar to these other markers(Figure 3). By the 14S stage, fch-expressing cells of thelateral plate have begun to migrate in a pattern similar to

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Figure 2

Protoporphyrin IX content of drcm248 mutant (fluorescent) andheterozygous or wild-type (non-fluorescent) embryos. Protoporphyrincontent is measured in µg/mg protein ± SD. Free protoporphyrin wasextracted with ethanol from 500 fluorescent and 500 non-fluorescentdrcm87 embryos at 3 dpf and assayed by HPLC. We confirmed that allof the porphyrin found by spectrofluorimetric assays (excitationwavelength = 402 nm; emission = 632 nm) was protoporphyrin by ourstandard HPLC assay [17].

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that of GATA1 and GATA2 and distinct from that ofangioblastic markers. This medial migration progresses tem-porally from anterior to posterior. By 24 hours developmentfch-expressing cells begin circulating, and it becomes clearthat the expression of fch is then restricted to erythrocytes.

Genetic and radiation hybrid mapping place the drc muta-tion and the fch gene within 0.1 centimorgans (cM) of eachother on linkage group 21 within a 3.4 cM interval definedby markers Z6295 and Z4718. drc is 1.2 cM from the closestmarker Z4718. Therefore, we isolated and sequenced fchcDNA and genomic DNA sequences from drc mutantembryos (Figure 4). We find that drcm248 has a point muta-tion, a G→T transversion, located in a 5′ splice donor

sequence, preventing the excision of intron sequence.Interestingly, this splice site is conserved in position withthe exon 4 splice donor site in the human fch gene [12],but no equivalent human mutation has been reported.The unexcised intron sequence begins with the stopcodon TAA, which is in-frame with the fch translation.Thus, the effect of the mutation is to cause prematurechain termination (91 amino acids instead of 409). Acryptic splice donor site located 27 base pairs (bp) down-stream in the unexcised intron sequence is used to splicethe mutant exon to the normal 3′ acceptor sequence sothat the cDNA of fch from drcm248 is 1432 bp, 27 bp longerthan the wild-type cDNA. Given that the first 55 aminoacids of fch are known to encode leader sequences for

Brief Communication 1003

Figure 3

Expression of fch in wild-type and mutantembryos. fch is expressed in the posteriorlateral plate mesoderm of embryos beforeerythropoietic precursors have migrated tothe midline as early as the 9 somite (S)stage. fch-expressing cells (brown) beginto migrate medially at 12–14S in a segmentalfashion, and form a wide single stripe at themidline. At 24 hpf, cells expressing fch arein the vein and artery, with the majority ofcells in the vein. No expression of fch isdetected in any other tissues up to 48 hpf.(a) 10S; (b) 14S; (c) 15S; (d) 17S; (e,f) 24hpf side and dorsal view. (g) An embryo at24 hpf in which circulation has begun showsfch expression in cells over the yolk in theducts of Cuvier. Anterior is to the left. For

the in situ hybridization, fch was digestedwith HindIII and transcribed from T7. The

probe was hybridized as previouslydescribed [18].

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Alignments of genomic DNA and cDNA fromthe fch gene of drcm248 and wild-typeembryos show a point mutation (arrow andboldface) at a splice donor site in genomicDNA, resulting in a 27 bp insertion of intronsequence into the cDNA. An in-frame stopcodon in the insertion in the intron sequence(underlined) truncates the predicted protein-coding sequence. A cryptic splice donorsequence 27 bp into the intron is used tosplice the cDNA to the correct downstreamexon, and thereby continue cDNAtranscription to the normal polyadenylationsite. The predicted protein sequence of fch indrcm248 is truncated with respect to the wild-type protein. Predicted binding sites for theiron-sulfur cluster in the wild-type protein areunderlined. The conservative amino-acidsubstitution at amino acid 84 is due to apolymorphism in the AB strain.

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m248 –/– TCATGCAGCTGCCCGTGCAAAATTAAGAAAGACTATTCAACATTTATAGGTCTAATATATACACTTCCTGAC|||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||

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cDNA Exon ExonIntron sequencem248 –/– TCATGCAGCTGCCCGTGCAAAATTAAGAAAGACTATTCAACATTTATAGTAAACTCGGGCCATTCATTGCCA

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TCATGCAGCTGCCCGTGCAAAA...........................TAAACTCGGGCCATTCATTGCCA

Predicted proteinm248 –/– MAVLGGACRLVQLVRCGSPVGLCLSSSLRRQSTATAAAFNTTATPETKESRKPKTGILML

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WTWT WTTMQGEGMVKLLDEMCPDTAPHKFYIGFRYVHPLTEEAIELMEKDGVERAVAFTQYPQY

WT SCSTTGSSLNAIYRYYSNRADRPKMRWSVIDRWPTHPLLIECFAEHVRNELDKFPVEKRD

WT DVVILFSAHSLPLSVVNRGDPYPQEVGATVQRVMDRLGHCNPYRLVWQSKVGPMAWLGPQ

WT TDEVIKGLCQRGKRNLLLVPIAFTSDHIETLHELDIEYSQVLGEEVGVENIRRAESLNGN

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PLFFRALADLVQSHLQSNESCSRQLTLRCPLCVNPTCAQTKAFFSSQKL 409

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NMGGPEKLEDVHDFLLRLFMDTDFMQLPVQNKLGPFIAKRRTPKIQEQYSKIGGGSPIKA

mitochondrial insertion, and are unconserved even amongclosely related species, we expect that the short peptide offch in drcm248 would be a functional null. In particular, it isknown that the 2Fe⋅2S cluster, which is encoded down-stream of the mutation in the carboxyl terminus of fch, isabsolutely necessary for the activity of vertebrate fer-rochelatases. This cluster would be completely absent indrcm248 ferrochelatase. A critical histidine residue wouldalso be missing in the truncated peptide [13].

Heme is a component of many essential cellular proteinsin addition to ferrochelatase. This suggests that someheme or ferrochelatase activity must be present duringearly embryonic stages, before heme synthesis begins inthe wild-type embyro. The survival of our apparently nullfch mutants to early larval stages also implies an earlysource of heme independent of ferrochelatase activity. Aswe have found no evidence of a second fch gene in thezebrafish genome, and, using the reverse transcription andpolymerase chain reaction (RT-PCR), did not find evi-dence for any residual correct splicing, we propose thatthere are maternally deposited stores of either heme, fchmRNA, or ferrochelatase protein in the yolk of theembryo. This is not unprecedented, as the zebrafish yolkis a store for the iron required for early hemoglobin syn-thesis [14], as well as for mRNAs that make a significantcontribution to early development [15,16].

This work emphasizes the potential relevance of genome-wide mutational analysis in zebrafish to heritable humandisease. As a model of EPP, the transparency and accessi-bility of the early zebrafish embryo permits studies of pro-toporphyrin-induced developmental organ toxicity undercontrolled light conditions. This ability can be exploitedto evaluate the light dependence of organotypic pheno-types, an approach complementary to studies in mice andtissue culture cells [3,5]. In the future, drc mutant zebrafishmight be used for direct in vivo screening to discoverchemical agents which might be useful for preventing orameliorating the symptoms of EPP in humans.

Supplementary materialSupplementary material including additional methodological detail isavailable at http://current-biology.com/supmat/supmatin.htm.

AcknowledgementsWe thank Nobu Shimoda and Galen Wo for help in running the MAP-MAKER program. We thank Richard W. Lambrecht for advice concerningassays for porphyrins. Grant support to M.C.F. is from R01RR0888,RO1DK55383, RO1HL49579, and a sponsored research agreement fromGenentech. Grant support to H.L.B. is from USPHS, NIH grant DK 38825.

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