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Prepublished online as a Blood First Edition Paper on August 22, 2002; DOI 10.1182/blood-2002-04-1169.
RED CELLS
From the Department of Hematology/Oncology, and Howard
Hughes Medical Institute, Children's Hospital, Boston MA; Institut de
Génétique et de Biologie Moléculaire et Cellulaire,
CNRS/Institut National de la Santé et de la Recherche
Médicale (INSERM)/ULP, BP 163, CU de Strasbourg 1, BP163, 67404 Illkirch Cedex, France; Department of
Medicine, University of Washington, Seattle; School of Biological
Sciences, Washington State University, Vancouver; Department of
Medicine, Washington University, St Louis MO; and Xenon Genetics,
Burnaby, BC, Canada.
Iron is an essential nutrient required for the function of all
cells, most notably for the production of hemoglobin in red blood
cells. Defects in the mechanisms of iron absorption, storage, or
utilization can lead to disorders of iron-limited erythropoiesis or
iron overload. In an effort to further understand these processes, we
have used the zebrafish as a genetic system to study vertebrate iron metabolism. Here we characterized the phenotype of
chardonnay (cdy), a zebrafish mutant
with hypochromic, microcytic anemia, and positioned the mutant gene on
linkage group 11. The cdy gene was isolated by a functional
genomics approach in which we used a combination of expression studies,
sequence analyses, and radiation hybrid panel mapping. We identified
erythroid-specific genes using a whole embryo mRNA in situ
hybridization screen and placed these genes on the zebrafish genomic
map. One of these genes encoded the iron transporter divalent metal
transporter 1 (DMT1) and colocalized with the cdy gene. We
identified a nonsense mutation in the cdy allele and
demonstrated that, whereas wild-type zebrafish DMT1 protein can
transport iron, the truncated protein expressed in cdy
mutants is not functional. Our studies further demonstrate the
conservation of iron metabolism in vertebrates and suggest the
existence of an alternative pathway of intestinal and red blood cell
iron uptake.
(Blood. 2002;100:4655-4659) Although iron is essential for the function of all
cells, in its free form it can be toxic. Vertebrates have evolved a
complex system to regulate the absorption, storage, and utilization of iron. According to the current model, iron enters the duodenal enterocyte via the transporter divalent metal transporter 1 (DMT1, formerly Nramp2/DCT1) with assistance from the ferric reductase duodenal cytochrome B (DCYTB).1-4 Iron is
transported into the circulation across the basolateral surface of the
enterocyte via the iron transporter ferroportin 1 (also known
as IREG1/MTP1), likely aided by the membrane-bound ferroxidase,
hephaestin.5-8 In mammals, the major pathway of absorption
of iron by cells involves uptake of transferrin-bound iron from the
serum via the transferrin receptor-mediated formation of endosomes.
The iron transporter DMT1 is required for the transport of iron out of
the endosome and into the cytosol, where it becomes available for
incorporation into newly synthesized proteins or for storage in
ferritin.9,10 Excess iron that is not required by the body
can be stored in the liver and in macrophages.
Disruption of the mechanisms of iron absorption, storage, and
utilization can lead to disease. Iron deficiency is the most common
cause of hypochromic, microcytic anemia in humans. Most cases are due
to inadequate dietary iron or blood loss; however, a rare fraction of
iron deficiency anemias appear to have a genetic component.11 In addition, the iron overload disorder
hemochromatosis is one of the most common genetic disorders in
individuals of northern European descent. The more rare disorders
aceruloplasminemia and atransferrinemia are also characterized by
tissue iron overload.12,13 Although presently no human
iron deficiency disorder has been associated with a mutation in a
particular gene, mutations in 3 different genes (Hfe, transferrin
receptor 2, and ferroportin 1) have been shown to cause different forms
of hemochromatosis.14-17
Animal models can help to further our understanding of iron metabolism.
For instance, the positional cloning of 2 naturally occurring rodent
mutants with hypochromic, microcytic anemia led to a greater
understanding of intestinal and erythrocyte iron absorption. Fleming
and colleagues showed that both the microcytic anemia mouse
(mk) and the Belgrade rat have a missense mutation in the
12-transmembrane domain iron transporter DMT1.4,9 Studies
of the Belgrade rat have demonstrated the critical role for DMT1 in 2 aspects of iron metabolism, namely, uptake of iron into the body from
the intestinal lumen and utilization of iron by cells via transferrin
receptor-mediated iron uptake, most notably by cells of the red blood
cell lineage.18-20
We have used the zebrafish as a genetic system to study vertebrate
blood development and iron metabolism. A large-scale ethylnitrosourea (ENU) mutagenesis screen identified 5 complementation groups of mutants
with an embryonic, hypochromic anemia: sauternes
(sau), zinfandel (zin),
weissherbst (weh), chardonnay
(cdy), and chianti (cia).21,22 We have previously
reported the positional cloning of 2 of these mutants, sau
and weh.5,23
The sau gene encodes aminolevulinic acid synthetase 2 (ALAS2), the first step in heme biosynthesis within the red cell
lineage. This provides an animal model of congenital sideroblastic
anemia. Our studies of the weh mutant led to the
identification of the iron exporter Fpn 1, a novel
gene required in the zebrafish embryo for absorption of
maternal iron and in the adult for intestinal iron absorption.
Additional studies of Fpn 1 in humans and mice support the
hypothesis that the iron export function of Fpn 1 is
involved in iron metabolism in multiple tissues, from placental and
intestinal iron absorption to iron recycling in
macrophages.5-7 Furthermore, patients with mutations in
Fpn 1 have a rare form of hemochromatosis.16,17
Based on studies of mk mice and Belgrade rats, we considered
the iron transporter DMT1 as a candidate for zebrafish mutants with
hypochromic anemia. Through a combination of positional and candidate
cloning approaches we identified zebrafish DMT1 as the gene mutated in
chardonnay animals. Here we describe the phenotypic characterization of the hypochromic, microcytic anemia of
chardonnay mutant embryos and adults. We cloned the
zebrafish homolog of DMT1 and characterized the expression pattern in
zebrafish embryos. In addition, we used genetic and physical mapping
techniques to demonstrate colocalization of the cdy gene
mutation and the dmt1 gene. Finally, we identified an A>T
mutation in the dmt1 gene of chardonnay animals
that leads to the formation of a stop codon distal to the sixth
transmembrane domain of the protein. Functional studies demonstrated
that this truncated DMT1 protein cannot transport iron. These studies
have identified the chardonnay mutant as an essential tool
for the study of iron metabolism in the zebrafish model system.
Phenotypic analysis and genetic and physical mapping
Genetic mapping strains were created by in vitro fertilization of the
eggs of cdy heterozygote AB females with the sperm of males
of polymorphic strains (DAR or SJD). Haploid and gynogenetic diploid
(early pressure technique) embryos were produced as
described.25 Linkage to centromeric simple sequence length
polymorphism (SSLP) markers by half-tetrad analysis was performed as
previously described.26,27 Haploid embryos from
cdyte216 AB/SJD or AB/DAR hybrid females were
genotyped with random amplified polymorphic DNA (RAPD) markers and SSLP
markers on zebrafish LG11.28,29
Amplified fragment length polymorphism (AFLP) marker identification was
performed on haploid embryos from a wehtp85c
AB/DAR hybrid female as previously described.5,30 AFLP
marker sequences were used to design polymerase chain reaction (PCR) primers for the identification of large insert genomic
clones.31 Forward and reverse primer sequences,
respectively: E5: 5'-GTAAGATAATGAAGCAGTTG-3' and
5'-AACAGCAAACCGAGTGTG-3'; E59: 5'-GAGCACCAGGAGACACTAGATG-3' and
5'-CTGCGTACCAATTCAGGGCTTC-3'; E54: 5'-TCGTCTGATCAGCCAAGTC-3' and
5'-TGAATGAAGTGAGTTTGAATG-3'; I22: 5'-GTCTCCAGAAACGTCCACAG-3' and
5'-TGTCTCTAATTTATAACGTACC-3'. Zebrafish yeast artificial
chromosome (YAC) clones were isolated by a PCR pooling strategy with
the above AFLP marker primers (Research Genetics, Huntsville,
AL).32 The bacterial artificial chromosome (BAC) clone
39J6 was isolated by hybridization of the LG11 RAPD marker 5G1400 to
filters representing the Genome Systems zebrafish BAC
library.23,33
The forward and reverse PCR primers for mapping markers onto the
radiation hybrid panel are as follows: RAPD marker 5G1400: 5'-GATTGTAGGATTTCAGTGTGTC-3' and 5'-CATCATCCAAACATCATCAGAA-3'; 39J6.SP6
(SP6 end of 39J6 BAC clone): 5'-ATCCGCATTTATTCAAGTCA-3' and
5'-CAACATTATTCGCCCTCCTG-3'; YAC 198D7 marker:
5'-CTGTTTGGCAGTGTATGATGC-3' and 5'-GTGCAGCGCTAGTGCTATTG-3';
dmt1 marker: 5'-GACACGACACACGCAGATCTCCAC-3' and
5'-GCCAATGGAGGAAGCAGAAGAATC-3'.34,35
Assay of iron uptake activity for wild-type and mutant DMT1
protein
A single autosomal recessive allele of the hypochromic
mutant chardonnay (cdyte216) was
identified as part of a large-scale screen for mutations in embryonic
development.22 Initial studies showed that homozygous mutant cdy animals survive to adulthood and appear similar
to their wild-type siblings. We characterized the red blood cells of
both cdy mutant embryos and adults. Although homozygous
mutant cdy embryos have a wild-type number of circulating
red blood cells up until 48 hours after fertilization (hpf),
hemoglobin expression was not detected at 30 and 48 hpf (Figure
1A). These results suggest that
cdy mutant embryos have a defect in the production of
hemoglobin molecules. This defect is not due to a lack of globin mRNA
expression, based on in situ hybridization for
We next analyzed the blood of cdy adults to determine if the genetic lesion also affected adult hematopoiesis. Wright-Giemsa staining demonstrates that the peripheral red blood cells of cdy mutants are abnormal compared to wild-type (Figure 1C, note the less condensed nuclei). In addition, both the peripheral blood and kidneys (adult site of hematopoiesis) of adult cdy mutants appear to have an increased number of red blood cell precursors (Figure 1C), consistent with increased erythropoiesis in response to anemia. Measurements of MCV and MCH in the peripheral blood of cdy mutant adults demonstrate that these animals have a hypochromic, microcytic anemia (Figure 1D). The first step in identifying the cdy mutant gene was
genetic mapping. We mapped the cdy locus to zebrafish
linkage group 11 by half-tetrad analysis with gynogenetic diploid
embryos.27 We used AFLP analysis to identify 4 markers
linked to the cdy mutation: E5, E54, E59, and I22 (Figure
2A).30,37 We subsequently isolated gDNA clones that contained the AFLP markers linked to cdy. This analysis identified a contiguous stretch of YAC
clones closely linked to the cdy locus (Figure 2A). As part
of a candidate cloning strategy, we positioned our
cdy-linked markers on the T51 radiation hybrid (RH) panel of
cell lines. These data can be visualized on the Zon laboratory
genomics Web site http://134.174.23.167/zonrhmapper/, as
CHUNP111(198D7), CHUNP113(39J6SP6end), and CHUNP115(5G1400). Initially, no obvious candidate genes for the cdy mutant
localized near these markers on the RH map.
We isolated the cdy gene using a functional genomics
approach. The expression of random genes from different embryonic cDNA libraries was systematically examined in zebrafish embryos from 6 different developmental stages: gastrula, neurula, tail elongation, 24 hpf, 36 hpf, and 48 hpf. We analyzed a total of 5414 cDNAs, among which
1326 (24%) showed spatially restricted expression patterns. Of the
spatially restricted genes, 41 (3%) were found to be expressed in
hematopoietic cells. One of the genes isolated in this screen was the
zebrafish homolog of the iron transporter DMT1. Zebrafish
dmt1 encodes a predicted protein of 547 amino acids with
73% identity to human and mouse DMT1 (Figure 2B). Zebrafish dmt1 mRNA is expressed in bilateral stripes of putative
blood precursors at the 4-somite stage of development (Figure
3A). Expression of dmt1 is
maintained in erythroid cells of the zebrafish blood island and in
circulating erythroid cells as late as day 3 of development (Figure
3B-D, and data not shown). Later in development expression appears in
the intestine (Figure 3E-F). Taken together, these expression domains
are consistent with the established role of DMT1 in iron metabolism in
higher vertebrates.
Based on the mammalian animal models of hypochromic anemia, we considered zebrafish dmt1 as a very strong candidate for zebrafish blood mutants with hypochromic anemia.22,38 As part of our candidate cloning strategy, we mapped dmt1 on the zebrafish RH map and demonstrated very close linkage of the dmt1 gene (CHUNP112) and the cdy-linked markers. Subsequent PCR analysis showed that dmt1 is contained on 3 YAC clones in the contig, 130B7, 198D7, and 159D2 (Figure 2A). Based on this close linkage, we cloned the cdyte216 mutant allele of dmt1 from embryo RNA. Sequence analysis showed that cdy mutants have an A>T nucleotide change that results in the formation of a stop codon (K264X) distal to sequence encoding the sixth transmembrane domain of the DMT1 protein (Figure 2B). The identification of a stop codon half-way into the dmt1 coding region, considered together with the previous identification of mouse and rat mutants in DMT1, provides substantial evidence that the hypochromia of cdy mutants is caused by this mutation in the dmt1 gene. We further characterized zebrafish DMT1 by analyzing its ability to
transport iron. Rat DMT1 has been shown to transport a range of
divalent cations, including iron, manganese, lead, zinc, copper, and
cadmium.2 We tested zebrafish DMT1 for iron transport activity in a functional assay.9,36 The human embryonic
kidney cell line 293T was transfected with constructs that expressed either wild-type DMT1 protein or truncated
DMT1te216. Two days after transfection, uptake of
55Fe was measured at pH 6.0. This analysis showed
that DMT1-expressing cells take up 9.6-fold more iron than control
transfected cells (Figure 4). In
contrast, the truncated DMT1te216 protein does not function
as an iron transporter (Figure 4).
Here we have demonstrated that the chardonnay (cdy) mutant gene encodes the zebrafish ortholog of the iron transporter DMT1. We characterized the phenotype of cdy mutants and showed that, like rodent mutants in DMT1, cdy animals are viable and have a hypochromic, microcytic anemia. We initially took a positional cloning approach toward isolating the cdy gene. The cdy mutation was mapped to zebrafish LG11 by half-tetrad analysis. Close genetic markers were identified using the AFLP technique. Subsequently, several AFLP markers tightly linked to the cdy locus were used to identify a YAC contig. A candidate zebrafish cDNA, dmt1 (formerly NRAMP2/DCT1), was independently isolated by an in situ screen of random cDNAs and was found to be both present on this physical contig and mutated in cdyte216 animals. Finally, we demonstrated that wild-type zebrafish DMT1 transports iron in a functional assay, whereas the cdy mutant allele is nonfunctional. Animals that have mutations in DMT1, cdy zebrafish, mk mice, and Belgrade rats, cannot take up enough iron to make sufficient levels of hemoglobin, resulting in hypochromic, microcytic anemia. Although it has been demonstrated that the G185R missense mutation in mk mice and Belgrade rats has dramatically decreased function in an iron uptake assay, it remains possible that even a low level of function by a missense mutated DMT1 could explain the viability of mk and Belgrade animals.9,36 Based on the severity of the zebrafish cdy truncation mutation, we propose that zebrafish have an alternate protein or pathway for absorption and utilization of iron and that this alternate mechanism could also exist in mammals. There are a variety of possible mechanisms, at the level of the enterocyte or erythroid cell, by which an alternate pathway may allow cdy mutant animals to survive without functional DMT1 protein. It is possible that the alternate gene or genes play a role in non-transferrin-bound iron (NTBI) uptake in erythroid cells, either through uptake of ferritin or through a separate iron uptake system.39,40 However, this may be unlikely because it appears that the transferrin cycle is essentially the sole iron uptake pathway for erythroid cells. This is inferred from the finding that the hypotransferrinemia (hpx) mouse, which has a splicing defect in the transferrin gene, is nonviable and can only be rescued by injection of wild-type transferrin.41-43 Rescue by transferrin injection is necessary despite the fact that hpx animals have high levels of non-transferrin-bound iron in the serum. Based on studies of hpx mice, it appears most likely that the so-called alternate pathway would play a similar role as DMT1 in the endosome and possibly the enterocyte. From the original large-scale mutagenensis screen for zebrafish mutants with anemia, the cdy gene is the second iron transporter to be cloned. We previously described the positional cloning of the weh mutant as the novel iron transporter gene Fpn 1.5 Both the DMT1 and ferroportin1 transporters are also important in mammalian disease, suggesting that the genes involved in iron metabolism are evolutionarily conserved in vertebrates.4,9,16,17 Our studies in zebrafish have identified the first DMT1 truncation mutation in an animal model. The viability of this null mutant is important because it defines the existence of a DMT1-independent pathway for iron to reach the erythron. The ability to efficiently perform large-scale mutagenesis screens is one of the many advantages of the zebrafish animal model. The ultimate advantage of this system for studying iron metabolism will be the ability to design new and innovative screens for mutants. Mutagenesis screens for phenotypic modifiers can identify genes either within the same molecular pathway as the gene of interest or within a parallel pathway. We propose that the zebrafish system could be used to identify the alternate mechanism by which cdy mutant animals obtain iron. For instance, the zebrafish mutant cdy could be used in an ENU mutagenesis screen for modifiers, either enhancers or suppressors, of the cdy phenotype. Specifically, enhancing mutations would eliminate the function of genes involved in an alternate iron uptake pathway, resulting in an increase in severity of the anemia of cdy mutant animals. This type of screen is likely to produce mutations in genes of interest to the study of iron metabolism. The current wealth of genomics resources available for the cloning of zebrafish mutant genes makes the identification of new genes via ongoing and future modifier screens a viable experimental approach to the study of vertebrate iron metabolism.
Submitted April 17, 2002; accepted July 21, 2002.
Prepublished online as Blood First Edition Paper, August 22, 2002; DOI 10.1182/blood-2002-04-1169.
Supported by National Institutes of Health grants 5R01 DK053298 and 5R01 RR015402.
L.I.Z. is an Investigator of the Howard Hughes Medical Institute.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Leonard I. Zon, Children's Hospital, 300 Longwood Ave, Enders 750, Boston, MA 02115; e-mail: zon{at}enders.tch.harvard.edu.
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H.-F. Lin, D. Traver, H. Zhu, K. Dooley, B. H. Paw, L. I. Zon, and R. I. Handin Analysis of thrombocyte development in CD41-GFP transgenic zebrafish Blood, December 1, 2005; 106(12): 3803 - 3810. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
- and 
-globins at
36 hpf (Figure 
