1. Volume 10, Issue 16, Pages 1001-1004 (August 2000)
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, Mark C. Fishman 
Current Biology 
Volume 10, Issue 16, Pages (August 2000)
DOI: /S (00)

2. Figure 1
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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3. Figure 2
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].
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4. 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 the in situ hybridization, fch was digested with HindIII and transcribed from T7. The probe was hybridized as previously described [18].
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5. 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 protein-coding 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 wild-type 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.
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