Ferroportin1 is required for normal iron cycling in zebrafish
Paula G. Fraenkel, … , David Zahrieh, Leonard I. Zon
Paula G. Fraenkel, … , David Zahrieh, Leonard I. Zon
Published June 1, 2005
Citation Information: J Clin Invest. 2005;115(6):1532-1541. https://doi.org/10.1172/JCI23780.
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Research Article Hematology

Ferroportin1 is required for normal iron cycling in zebrafish

Abstract

Missense mutations in ferroportin1 (fpn1), an intestinal and macrophage iron exporter, have been identified between transmembrane helices 3 and 4 in the zebrafish anemia mutant weissherbst (wehTp85c–/–) and in patients with type 4 hemochromatosis. To explore the effects of fpn1 mutation on blood development and iron homeostasis in the adult zebrafish, wehTp85c–/– zebrafish were rescued by injection with iron dextran and studied in comparison with injected and uninjected WT zebrafish and heterozygotes. Although iron deposition was observed in all iron-injected fish, only wehTp85c–/– zebrafish exhibited iron accumulation in the intestinal epithelium compatible with a block in iron export. Iron injections initially reversed the anemia. However, 8 months after iron injections were discontinued, wehTp85c–/– zebrafish developed hypochromic anemia and impaired erythroid maturation despite the persistence of iron-loaded macrophages and elevated hepatic nonheme iron stores. Quantitative real-time RT-PCR revealed a significant decrease in mean hepatic transcript levels of the secreted iron-regulator hepcidin and increased intestinal expression of fpn1 in anemic wehTp85c–/– adults. Injection of iron dextran into WT or mutant zebrafish embryos, however, resulted in significant increases in hepcidin expression 18 hours after injection, demonstrating that hepcidin expression in zebrafish is iron responsive and independent of fpn1’s function as an iron exporter.

Authors

Paula G. Fraenkel, David Traver, Adriana Donovan, David Zahrieh, Leonard I. Zon

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Quantification of transcript levels by multiplex real-time PCR. RNA from...
Quantification of transcript levels by multiplex real-time PCR. RNA from liver (A, B, and D) or intestine (C) of 10- to 12-month-old adult zebrafish or pools of embryos (E and F) was used to generate cDNA templates for multiplex reactions amplifying the gene of interest and β-actin. Transcript abundance, normalized to β-actin expression, is expressed as a fold increase over a calibrator sample. (A–D) Expression of transferrin (A), fpn1 (B and C), or hepcidin (D) in adult WT, heterozygote, or mutant zebrafish treated with iron 6–8 months previously (+Fe) or never treated (no Fe). The calibrator sample was an uninjected WT adult; n = 6–9 individuals per cohort. *P = 0.01 compared with iron-injected WT zebrafish. **P < 0.0001 compared with iron-injected WT zebrafish. (E and F) At 48 hours after fertilization, embryos were anesthetized and injected with iron dextran (+) or anesthetized without iron injection (–). (E) Eighteen-hour time course of hepcidin transcript abundance following iron dextran injection. The calibrator sample consisted of a pool of embryos 2 hours after iron injection; n = 2 pools of embryos per cohort. ‡P < 0.05 compared with uninjected age-matched embryos. (F) Induction of hepcidin expression 18 hours after iron dextran injection of wehTp85c–/– or WT sibling embryos. The calibrator sample was an uninjected pool of embryos; n = 3 pools of embryos per cohort. ‡‡P < 0.01 compared with uninjected age-matched embryos.