1532 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 6 June 2005
Ferroportin1 is required for normal
iron cycling in zebrafish
Paula G. Fraenkel,1,2 David Traver,1 Adriana Donovan,1 David Zahrieh,3 and Leonard I. Zon1
1Division of Hematology/Oncology, Children’s Hospital, Karp Research Laboratories, Boston, Massachusetts, USA.
2Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
3Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.
Missense mutations in ferroportin1 (fpn1), an intestinal and macrophage iron exporter, have been identi-
fied 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 ane-
mia. 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 sig-
nificant 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.
According to a current model of iron transport (1), duodenal
enterocytes and placental syncytiotrophoblasts are the principle
cells responsible for iron acquisition in mammals. At the apical
surface of the duodenal enterocyte, the divalent metal transporter 1
(DMT1) mediates uptake of iron from the intestinal lumen into
the enterocyte (2, 3). Absorbed iron may be stored bound to ferritin
or exported across the enterocyte’s basolateral membrane to the
circulation via ferroportin1 (fpn1) (4). In the plasma, iron binds
transferrin and is subsequently imported into erythroid precur-
sors and other cells via transferrin-receptor–mediated endocytosis.
Erythrocytes have no known means of iron export but rather incor-
porate iron into hemoglobin. Macrophages serve as iron scaven-
gers, phagocytosing senescent erythrocytes, degrading hemoglobin,
and storing iron incorporated in ferritin.
Hepcidin, a small cysteine-rich peptide with antimicrobial prop-
erties (5, 6), has recently been identified as a key regulator of iron
absorption and utilization. Produced in the liver, hepcidin cir-
culates in plasma and is excreted in the urine. Hepcidin protein
levels appear to be largely regulated by transcriptional control
(1). Hepcidin null mice develop iron overload (7) while animals
that overexpress hepcidin develop severe hypochromic anemia (8).
Hepatic expression of the hepcidin gene decreases in response to
iron deficiency, hypoxia, and anemia (9) while expression increases
in response to iron overload (10) or inflammation (9, 11).
The zebrafish, Danio rerio, provides an excellent system for the
identification and analysis of genes involved in iron metabolism and
erythroid development. Analogous to higher vertebrates, zebrafish
exhibit multilineage hematopoiesis, resulting in erythroid, mono-
cyte, granulocyte, and thrombocyte lineages, and undergo hemoglo-
bin switching (12). Large-scale genetic screens for embryonic hypo-
chromic anemia have identified recessive mutations with defects in
heme synthesis (13), globin production (12), and iron acquisition
via DMT1 (14) and transferrin receptor (15). Positional cloning of
the mutation responsible for the hypochromic zebrafish mutant
weissherbst (weh) resulted in the identification of the basolateral iron
exporter, fpn1, (4) also known as IREG1 (16) and MTP1 (17), a con-
served protein with 10 putative transmembrane domains and a func-
tional iron response element (18) in the 5′ untranslated region.
Two alleles of the weh phenotype have been described previous-
ly (19). Both have a homozygous recessive pattern of inheritance.
The wehTh238 allele has a premature stop mutation and is thought
to be a null allele (4). The wehTp85c allele is of particular inter-
est because it encodes a missense mutation (Leu 167 to Phe) in
the same conserved region of fpn1 (4) as several of the missense
mutations identified in patients with type 4 hemochromatosis
(20–26). Both wehTp85c and wehTh238 homozygotes develop severe
anemia by 48 hours after fertilization and die between 10 and 14
days of age. A single intravenous or intramuscular iron dextran
injection allowed the homozygote embryos to survive (4) for sev-
eral weeks but not to reach maturity.
Here we demonstrate that administration of multiple iron injec-
tions enabled the wehTp85c–/– zebrafish to survive to adulthood,
facilitating evaluation of the effects of the fpn1 mutation on blood
development and iron homeostasis. We provide evidence that
wehTp85c–/– adults have impaired iron export, both from enterocytes
and macrophages, and that normal fpn1 function is not required
for iron-responsive regulation of hepcidin in the zebrafish.
Nonstandard abbreviations used: CH, cellular hemoglobin; DAB, diaminobenzi-
dine; DMT1, divalent metal transporter 1; FSC, forward scatter; fpn1, ferroportin1;
HFE, hemochromatosis gene product; IRE, iron regulatory element; MCV, mean
corpuscular volume; pcm, polycythemia; SSC, side scatter; wehTp85c–/–, zebrafish anemia
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J. Clin. Invest. 115:1532–1541 (2005).
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 6 June 2005
The effects of a missense mutation in fpn1 have not been evaluated
previously in an adult model organism. In order to study the effects
of fpn1 deficiency on erythroid development and iron homeostasis,
we rescued wehTp85c homozygotes to adulthood by administering
iron dextran injections early in development. Without iron injec-
tions, 0 of 529 homozygous mutants were alive at 3 weeks. Treat-
ment with a single iron injection at 72 hours rescued the anemia
phenotype and allowed 47 of 87 (54%) of the mutants (wehTp85c–/–)
to survive to 1 month, which was similar to the survival rate of iron-
injected (37 of 61; 61%) and uninjected (47 of 89; 53%) controls
(wehTp85c+/– and wehTp85c+/+). Without additional iron injections, the
mutant animals did not continue to mature. After a total of 4 injec-
tions of iron dextran were administered to the wehTp85c–/– zebrafish
(day 3, week 5, week 8, and week 16), the animals reached adult size.
This regimen was adopted for subsequent experiments.
As fpn1 has been shown to be expressed in the yolk syncytial layer
that separates the embryo from the iron-rich yolk, we postulated
that the weh mutation could have a maternal-dominant effect,
impairing export of iron to the embryo from maternally derived
stores. WehTp85c homozygous females crossed with wehTp85c+/+ males
produced only apparently normal, nonanemic embryos (data not
shown). This indicates that iron transfer into the zebrafish embryo
does not depend on maternal transcripts of fpn1. Either fpn1 tran-
scripts derived from the zygote are sufficient, or iron is transferred
into the embryo via an fpn1-independent mechanism.
The wehTp85c homozygotes and controls were monitored as they
aged. At 6 months of age, 2 months after the last iron injection,
wehTp85c–/– did not differ in appearance from injected or uninjected
controls. By 12 months of age, however, the homozygote animals
had developed pallor (Figure 1A), and the peripheral blood was
hypochromic on a blood smear (Figure 1B). To assess the severity of
anemia, we measured mean corpuscular volume (MCV) (Figure 1C)
and cellular hemoglobin (CH) (Figure 1D) from the peripheral
blood of individual zebrafish. MCV is a measure of erythrocyte size
while CH indicates the amount of hemoglobin per erythrocyte. At 6
months of age, there was no significant difference in MCV and CH
among the cohorts. The MCV was 81.8 ± 9.82 fl for wehTp85c–/– zebra-
fish versus 99.2 ± 6.65 for iron-injected WT zebrafish (P = 0.26). CH
was 26.6 ± 3.4 pg for wehTp85c–/– zebrafish versus 29.9 ± 1.74 pg for
iron-injected WT zebrafish (P = 0.50). When the zebrafish reached
12 months of age, however, we observed a reduction in MCV in iron-
injected wehTp85c–/– zebrafish compared with iron-injected WT zebra-
fish (69.6 ± 1.43 fl vs. 95.4 ± 1.36 fl, respectively; P < 0.0001) or unin-
jected WT zebrafish (89.9 ± 2.93 fl; P = 0.0008). Similarly, the amount
of hemoglobin per erythrocyte (CH) was reduced significantly from
32.2 ± 2.53 pg in iron-injected WT zebrafish to 23.4 ± 0.78 pg in iron-
injected wehTp85c–/– zebrafish (P < 0.0001). The decrease observed in
both erythrocyte size and hemoglobin content are consistent with
anemia due to impaired hemoglobin production.
To examine the effect of fpn1 deficiency on the adult site of
zebrafish hematopoiesis, kidneys from individual 12-month-old
wehTp85c–/– zebrafish and controls were dissected. Flow cytometry was
used to separate the kidney marrow cells into 4 populations based
on differences in forward scatter (FSC) and side scatter (SSC) in
controls (Figure 2A) and wehTp85c–/– zebrafish (Figure 2B). The mean
percentages of cells in these populations were compared (Figure 2C).
This revealed a 3-fold reduction in the mean percentage of mature
erythrocytes in wehTp85c–/– zebrafish (11.8 ± 1.10 in wehTp85c–/– vs.
36.8 ± 1.93 in WT plus iron; P < 0.0001). Also observed was a doubling
The phenotype of the weh mutation in adult zebrafish. (A) Photographs of an iron-injected WT zebrafish (left) compared with a pale, iron-injected
homozygote (wehTp85c–/–) (right) at 1 year of age. (B) Erythrocytes from peripheral blood of iron-injected wehTp85c–/– (Mut + Fe; right) at 1 year of
age exhibit pale cytoplasm and decondensed nuclei compared with WT zebrafish (WT no Fe; left) stained with Wright-Giemsa. Magnification
×100. Scale bars: 10 microns. (C and D) Erythroid indices were obtained with an ADVIA 120 automated analyzer at 6 months (gray bars, n = 3–5)
and 12 months (white bars, n = 4–6) for each cohort: uninjected wehTp85c+/+ zebrafish (WT no Fe), iron-injected wehTp85c+/+ zebrafish (WT + Fe),
uninjected wehTp85c+/– zebrafish (Het no Fe), iron-injected wehTp85c+/– zebrafish (Het + Fe), and iron-injected wehTp85c–/– zebrafish (Mut + Fe). MCV
(C) is a measure of erythrocyte size, while CH (D) quantitates the amount of hemoglobin per erythrocyte. Data shown are means ± SE. ND, not
done. *P < 0.0001 compared with 12-month-old iron-injected WT zebrafish.
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 6 June 2005
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