Zebrafish dracula encodes ferrochelatase and its mutation provides a model for erythropoietic protoporphyria

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Zebrafish dracula encodes ferrochelatase and its mutation provides a model for erythropoietic protoporphyria Sarah Childs*†, Brant M. Weinstein*†‡, Manzoor-Ali P.K. Mohideen*§, Susan Donohue*¶, Herbert Bonkovsky*¶ and Mark C. Fishman* Exposure to light precipitates the symptoms of several genetic disorders that affect both skin and internal organs. It is presumed that damage to non-cutaneous organs is initiated indirectly by light, but this is difficult to study in mammals. Zebrafish have an essentially transparent periderm for the first days of development. In a previous large-scale genetic screen we isolated a mutation, dracula (drc), which manifested as a lightdependent lysis of red blood cells [1]. We report here that protoporphyrin IX accumulates in the mutant embryos, suggesting a deficiency in the activity of ferrochelatase, the terminal enzyme in the pathway for heme biosynthesis. We find that homozygous drcm248 →T transversion at a splice mutant embryos have a G→ donor site in the ferrochelatase gene, creating a premature stop codon. The mutant phenotype, which shows light-dependent hemolysis and liver disease, is similar to that seen in humans with erythropoietic protoporphyria, a disorder of ferrochelatase.

under normal light, and drcm159 has only a few circulating cells at that time [1]. drc erythrocytes are strongly fluorescent when exposed to epifluorescent illumination using a rhodamine filter (peak illumination at 510–560 nm). Upon illumination, red cells immediately begin to lyse within the blood vessels, and completely disappear within 2 minutes (Figure 1a). Under standard microscopic white light illumination, the lysis proceeds more slowly, and takes 15–20 minutes to be complete. Rapid erythrocyte lysis has other detrimental effects upon the embryo, manifest as bradycardia and diminished cardiac contractility. This appears to depend on the acuity of hemolysis, as embyros of Figure 1

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Received: 10 April 2000 Revised: 16 June 2000 Accepted: 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 discussion In a large-scale mutant screen we identified several classes of recessive lethal mutations with reduced numbers of or no red blood cells [1]. One subset, including drc, exhibits a far more pronounced anemia when raised under standard ambient light conditions than if raised in the dark. We isolated four alleles of drc: drcm87, drcm248 and drcm328 lack blood entirely four days post-fertilization (4 dpf) when raised

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Correspondence: Mark C. Fishman E-mail: [email protected]

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Address: *Cardiovascular Research Center, Massachusetts General Hospital East, Charlestown, Massachusetts 02129, USA. of Molecular Genetics, National Institute Present addresses: of Child Health and Development, Maryland 20892, USA. §Jake Gittlen Cancer Research Institute, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA. ¶The Center for Study of Disorders of Iron and Porphyrin Metabolism and The Division of Digestive Disease and Nutrition, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.

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Blood and liver phenotypes of drc mutant zebrafish. (a) Time-lapse series showing autofluorescent erythrocytes in the trunk of 1 dpf drcm248 embryos visualized under fluorescent light with a rhodamine filter (peak illumination at 510–560 nm). Circulation was arrested by use of Tricaine. By 120 sec, all erythrocytes have lysed. (b) Side view of 6 dpf drcm248 mutant and wild-type (WT) larvae under white light (top panels) and fluorescent light (bottom panels). The reddish liver in the mutants has a large number of red-brown inclusions that autofluoresce. (c) Cross-sections of 6 dpf drcm248 mutant and wild-type embryos under fluorescent (left) and Nomarski (right) optics. Mutants show extensive autofluorescence in the liver, and pronephric ducts but not in other 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 coverslips and 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 from birth have a similar degree of anemia, but without abnormal heart rate or contractility. The liver of drcm248 mutant embryos shows several hepatic abnormalities. It has a red hue and many inclusions are visible under both white and fluorescent light (Figure 1b). The cells are pleiomorphic and show variable histological atypia (data not shown). The hepatic inclusions are not limited to the region of vessels, but are scattered throughout the parenchyma (Figure 1c). In drcm248, these hepatic inclusions accumulate even in dark-raised embryos. We also observed fluorescent inclusions in the pronephric ducts of 6 dpf drcm248 embryos. At 4 dpf, the zebrafish kidney becomes a major site of erythropoiesis, and thus the accumulation of fluorescence there is likely to be due to the presence of erythrocytes in the tissue. The phenotype of drc mutants is suggestive of a group of disorders known as porphyrias in humans, which are caused by mutations in enzymes of the heme biosynthetic pathway. The sequential actions of eight enzymes generate heme, a pathway initiated by condensation of glycine and succinyl CoA into δ-aminolevulinate. Defects in the last five enzymes of the pathway are accompanied by accumulation of porphyrin intermediates. Illumination of these highly light-sensitive compounds causes the production of singlet oxygen and free radicals, leading, among other effects, to lipid peroxidation [2,3]. Patients show a constellation of symptoms which vary among porphyrias. They include cutaneous sensitivity to light, hemolysis of autofluorescent Figure 2

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red blood cells and hepatic pathology. A phenotype similar to that of drc has been observed in the zebrafish mutation yquem, which has a mutation in uroporphyrinogen decarboxylase, an enzyme in this pathway [4]. drc is, by complementation analysis, a different gene from yquem. We examined porphyrin levels in drcm87 embryos by HPLC. One porphyrin in particular, protoporphyrin IX, is present at very high levels in drc mutant embryos (Figure 2) whereas it is nearly undetectable in a pool of wild-type and heterozygous embryos. This suggested that the mutation in drc mutant embyros might lie in the ferrochelatase (fch) gene, which encodes an enzyme that converts protoporphyrin IX into heme by catalyzing the transfer of iron to the heme moiety. In humans, mutations in fch cause the disorder erythropoietic protoporphyria (EPP; OMIM entry 177000). Patients have autofluorescent, light-sensitive red cells, and some develop severe liver disease. In humans, red cells are thought to be exposed to light during their transit through the skin, and it is presumed that the liver is damaged by uptake of toxic substances released by their lysis, as well as from local synthesis of protoporphyrin [5]. Crystalline deposits of protoporphyrin have been observed in the livers of patients with EPP [6]. The inclusions we observe in the liver of 6 day drc mutants may be similar to these, although they appear not to be biorefringent, and may represent a stage before the formation of large crystals. To determine whether fch is mutated in drc we first cloned the wild-type zebrafish gene. The cDNA is 1405 bp long and encodes a putative 409-amino-acid protein (GenBank accession number AF250368). The predicted protein sequence is highly conserved through evolution, with a >80% identity to human and other vertebrate ferrochelatases, 57% identity to the Drosophila gene, and 50% identity to the Saccharomyces cerevisiae gene.

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Protoporphyrin IX content of drcm248 mutant (fluorescent) and heterozygous or wild-type (non-fluorescent) embryos. Protoporphyrin content is measured in µg/mg protein ± SD. Free protoporphyrin was extracted with ethanol from 500 fluorescent and 500 non-fluorescent drcm87 embryos at 3 dpf and assayed by HPLC. We confirmed that all of the porphyrin found by spectrofluorimetric assays (excitation wavelength = 402 nm; emission = 632 nm) was protoporphyrin by our standard HPLC assay [17].

In situ hybridization shows that expression of fch in the lateral plate mesoderm begins early in development, around the 9–10 somite (S) stages. This is later than the early hematopoietic markers GATA2 and GATA1 [7], but contemporaneous with other markers of differentiated erythrocytes, such as aminolevulinate synthase (ALAS) [8]. At this time, cells which will adopt a hematopoietic fate are interspersed in the lateral plate with cells which will become vessel and pronephric duct. In fact, there may be a shared common precursor cell for the first intra-embryonic blood and angioblasts, a cell tentatively called the hemangioblast [9]. flk-1, fli-1 and scl are expressed by both hematopoietic and angioblast lineages [10,11]. At the 9–10S stage, fch is expressed in the trunk (but not the head) of the zebrafish embryo in a pattern similar to these other markers (Figure 3). By the 14S stage, fch-expressing cells of the lateral plate have begun to migrate in a pattern similar to

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Figure 3 Expression of fch in wild-type and mutant embryos. fch is expressed in the posterior lateral plate mesoderm of embryos before erythropoietic precursors have migrated to the midline as early as the 9 somite (S) stage. fch-expressing cells (brown) begin to migrate medially at 12–14S in a segmental fashion, and form a wide single stripe at the midline. At 24 hpf, cells expressing fch are in the vein and artery, with the majority of cells in the vein. No expression of fch is detected in any other tissues up to 48 hpf. (a) 10S; (b) 14S; (c) 15S; (d) 17S; (e,f) 24 hpf side and dorsal view. (g) An embryo at 24 hpf in which circulation has begun shows fch expression in cells over the yolk in the ducts of Cuvier. Anterior is to the left. For

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

that of GATA1 and GATA2 and distinct from that of angioblastic markers. This medial migration progresses temporally from anterior to posterior. By 24 hours development fch-expressing cells begin circulating, and it becomes clear that the expression of fch is then restricted to erythrocytes. Genetic and radiation hybrid mapping place the drc mutation and the fch gene within 0.1 centimorgans (cM) of each other on linkage group 21 within a 3.4 cM interval defined by markers Z6295 and Z4718. drc is 1.2 cM from the closest marker Z4718. Therefore, we isolated and sequenced fch cDNA and genomic DNA sequences from drc mutant embryos (Figure 4). We find that drcm248 has a point mutation, a G→T transversion, located in a 5′ splice donor

probe was hybridized as previously described [18].

sequence, preventing the excision of intron sequence. Interestingly, this splice site is conserved in position with the 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 stop codon TAA, which is in-frame with the fch translation. Thus, the effect of the mutation is to cause premature chain termination (91 amino acids instead of 409). A cryptic splice donor site located 27 base pairs (bp) downstream in the unexcised intron sequence is used to splice the mutant exon to the normal 3′ acceptor sequence so that the cDNA of fch from drcm248 is 1432 bp, 27 bp longer than the wild-type cDNA. Given that the first 55 amino acids of fch are known to encode leader sequences for

Figure 4 Alignments of genomic DNA and cDNA from the fch gene of drcm248 and wild-type embryos show a point mutation (arrow and boldface) at a splice donor site in genomic DNA, resulting in a 27 bp insertion of intron sequence into the cDNA. An in-frame stop codon in the insertion in the intron sequence (underlined) truncates the predicted proteincoding sequence. A cryptic splice donor sequence 27 bp into the intron is used to splice the cDNA to the correct downstream exon, and thereby continue cDNA transcription to the normal polyadenylation site. The predicted protein sequence of fch in drcm248 is truncated with respect to the wildtype protein. Predicted binding sites for the iron-sulfur cluster in the wild-type protein are underlined. The conservative amino-acid substitution at amino acid 84 is due to a polymorphism in the AB strain.

Mutation Genomic DNA m248 –/– TCATGCAGCTGCCCGTGCAAAATTAAGAAAGACTATTCAACATTTATAGGTCTAATATATACACTTCCTGAC |||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||| TCATGCAGCTGCCCGTGCAAAAGTAAGAAAGACTATTCAACATTTATAGGTCTAATATATACACTTCCTGAC

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TCATGCAGCTGCCCGTGCAAAATTAAGAAAGACTATTCAACATTTATAGTAAACTCGGGCCATTCATTGCCA |||||||||||||||||||||| ||||||||||||||||||||||| TCATGCAGCTGCCCGTGCAAAA...........................TAAACTCGGGCCATTCATTGCCA

Predicted protein m248 –/– MAVLGGACRLVQLVRCGSPVGLCLSSSLRRQSTATAAAFNTTATPETKESRKPKTGILML WT m248 WT WT WT WT WT WT

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|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| MAVLGGACRLVQLVRCGSPVGLCLSSSLRRQSTATAAAFNTTATPETKESRKPKTGILML NMGGPEKLEDVHDFLLRLFMDTDLMQLPVQN* ||||||||||||||||||||||| ||||||| NMGGPEKLEDVHDFLLRLFMDTDFMQLPVQNKLGPFIAKRRTPKIQEQYSKIGGGSPIKA WTTMQGEGMVKLLDEMCPDTAPHKFYIGFRYVHPLTEEAIELMEKDGVERAVAFTQYPQY SCSTTGSSLNAIYRYYSNRADRPKMRWSVIDRWPTHPLLIECFAEHVRNELDKFPVEKRD DVVILFSAHSLPLSVVNRGDPYPQEVGATVQRVMDRLGHCNPYRLVWQSKVGPMAWLGPQ TDEVIKGLCQRGKRNLLLVPIAFTSDHIETLHELDIEYSQVLGEEVGVENIRRAESLNGN PLFFRALADLVQSHLQSNESCSRQLTLRCPLCVNPTCAQTKAFFSSQKL

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mitochondrial insertion, and are unconserved even among closely related species, we expect that the short peptide of fch in drcm248 would be a functional null. In particular, it is known that the 2Fe⋅2S cluster, which is encoded downstream of the mutation in the carboxyl terminus of fch, is absolutely necessary for the activity of vertebrate ferrochelatases. This cluster would be completely absent in drcm248 ferrochelatase. A critical histidine residue would also be missing in the truncated peptide [13]. Heme is a component of many essential cellular proteins in addition to ferrochelatase. This suggests that some heme or ferrochelatase activity must be present during early embryonic stages, before heme synthesis begins in the wild-type embyro. The survival of our apparently null fch mutants to early larval stages also implies an early source of heme independent of ferrochelatase activity. As we have found no evidence of a second fch gene in the zebrafish genome, and, using the reverse transcription and polymerase chain reaction (RT-PCR), did not find evidence for any residual correct splicing, we propose that there are maternally deposited stores of either heme, fch mRNA, or ferrochelatase protein in the yolk of the embryo. This is not unprecedented, as the zebrafish yolk is a store for the iron required for early hemoglobin synthesis [14], as well as for mRNAs that make a significant contribution to early development [15,16]. This work emphasizes the potential relevance of genomewide mutational analysis in zebrafish to heritable human disease. As a model of EPP, the transparency and accessibility of the early zebrafish embryo permits studies of protoporphyrin-induced developmental organ toxicity under controlled light conditions. This ability can be exploited to evaluate the light dependence of organotypic phenotypes, an approach complementary to studies in mice and tissue culture cells [3,5]. In the future, drc mutant zebrafish might be used for direct in vivo screening to discover chemical agents which might be useful for preventing or ameliorating the symptoms of EPP in humans. Supplementary material Supplementary material including additional methodological detail is available at http://current-biology.com/supmat/supmatin.htm.

Acknowledgements We thank Nobu Shimoda and Galen Wo for help in running the MAPMAKER program. We thank Richard W. Lambrecht for advice concerning assays for porphyrins. Grant support to M.C.F. is from R01RR0888, RO1DK55383, RO1HL49579, and a sponsored research agreement from Genentech. Grant support to H.L.B. is from USPHS, NIH grant DK 38825.

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