Department of Hematology/Oncology, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA
Author for correspondence (e-mail:
zon{at}enders.tch.harvard.edu)
Accepted 20 October 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Zebrafish, Hematopoiesis, Transferrin receptor, Iron, Gene duplication
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tfr1 is highly expressed on differentiating erythrocytes,
reflecting their substantial iron requirement to support hemoglobin synthesis
(Ponka and Lok, 1999). The
murine knockout of Tfr1 established that it was required for
erythropoiesis and embryonic development
(Levy et al., 1999
). Mice
homozygous for a null Tfr1 allele died of anemia before embryonic day
(E) 12.5, and displayed marked growth retardation, edema and signs of tissue
necrosis. Tfr1/ mice also showed neurologic
abnormalities, including kinking of their neural tubes and increased neuronal
apoptosis. In addition, Tfr1+/ embryos evinced
hypochromic, microcytic erythrocytes, consistent with iron deficiency.
Analysis of chimeric mice generated with
Tfr1/ embryonic stem cells illustrated that
Tfr1 was required postnatally for adult erythropoiesis and lymphopoiesis, as
Tfr1/ cells did not contribute to the bone
marrow, spleen or thymus (Ned et al.,
2003
). Conversely, Tfr1/ cells did
incorporate into all non-hematopoietic tissues, indicating that other pathways
of iron uptake were sufficient to permit their survival
(Ned et al., 2003
).
In addition to Tfr1, mammals also possess transferrin receptor 2 (Tfr2), a
type II membrane protein similar to Tfr1. Tfr2 is highly expressed in liver
hepatocytes and erythroid precursor cells, and can facilitate Tf-bound iron
entry in vitro, but its function remains poorly understood
(Fleming et al., 2000;
Fleming et al., 2002
;
Kawabata et al., 1999
;
Kawabata et al., 2001
;
Trinder and Baker, 2003
).
Furthermore, while alternative mechanisms to Tf/Tfr1-mediated iron acquisition
have been characterized in a variety of mammalian cell lines, their in-vivo
roles remain to be elucidated. These include direct uptake of non-Tf-bound
iron (NTBI) (Baker et al.,
1998
; Goto et al.,
1983
; Hodgson et al.,
1995
; Inman and
Wessling-Resnick, 1993
; Kaplan
et al., 1991
; Sturrock et al.,
1990
), Tfr1-independent uptake of Tf-bound iron
(Chan et al., 1992
;
Thorstensen et al., 1995
), and
receptor-mediated uptake of ferritin
(Gelvan et al., 1996
;
Konijn et al., 1994
;
Leimberg et al., 2003
;
Meyron-Holtz et al., 1999
).
Recently, the soluble protein 24p3/neutrophil gelatinase-associated lipocalin
(Ngal) was found to deliver iron to developing mammalian kidney epithelial
cells, with a pattern of cell binding and intracellular trafficking
independent from that of the Tf/Tfr1, and may present one avenue for the
cellular distribution of NTBI (Yang et
al., 2002
).
The inability of erythrocytes to obtain adequate iron for hemoglobin
synthesis, as well as defects in heme or globin production, causes
hypochromic, microcytic anemias in humans. While low dietary iron or blood
loss is most frequently the underlying cause, inherited mutations in any
number of genes required for hemoglobin synthesis have been attributed to such
anemias (Andrews, 1999). We
have utilized the zebrafish Danio rerio as a genetic model to study
hemoglobin production during vertebrate erythropoiesis
(Brownlie and Zon, 1999
;
Wingert and Zon, 2003
).
Zebrafish hematopoietic screens have resulted in the identification of nine
complementation groups that display hypochromic, microcytic anemia:
chardonnay, chianti, frascati, gavi, montalcino, sauternes, shiraz,
weissherbst and zinfandel (K.D., P.F., R.A.W. and L.I.Z.,
unpublished) (Haffter et al.,
1996
; Ransom et al.,
1996
). chardonnay (cdy) has a mutation in
Dmt1 (Donovan et al.,
2002
). The weissherbst (weh) mutant is unable to
obtain maternal iron yolk stores due to a defect in ferroportin 1
(Fpn1), a transmembrane protein required to transport iron from the yolk
into embryonic circulation (Donovan et
al., 2000
). The mutant sauternes (sau) has a
defect in the enzyme aminolevulinate synthase-2 (Alas2), which functions at
the first step in heme biosynthesis
(Brownlie et al., 1998
).
Lastly, the zinfandel (zin) mutation has been mapped to the
major globin locus, suggesting that zin results from disrupted globin
function (Brownlie et al.,
2003
).
We report here the characterization of the chianti (cia) mutant phenotype and the cloning of the cia gene. We show that cia encodes an erythroid-specific isoform of transferrin receptor 1 (tfr1a) that is solely required for iron acquisition by differentiating erythrocytes. We found that zebrafish have undergone and retained a duplication of Tfr1 during teleost evolution, adding tfr1a and tfr1b to the growing list of gene duplicates in teleosts. To determine the function of zebrafish tfr1b, we utilized a morpholino knockdown approach and found that tfr1b is not required for erythropoiesis, but rather necessary for normal development of non-hematopoietic cells. These findings establish that the combined functions of tfr1a and tfr1b in zebrafish embryos recapitulate the role of mammalian Tfr1. Thus the cia mutant provides a useful genetic model to study the role of Tfr1 in erythropoiesis in the absence of other developmental defects.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
o-dianisidine staining, in-situ hybridization and histological analysis
Detection of hemoglobin by o-dianisidine was performed as
described (Ransom et al.,
1996). We performed whole-mount in-situ hybridization with
digoxigenin-labeled RNA probes as described
(Thompson et al., 1998
).
Synthesis of ße1 globin probe was performed as described
(Brownlie et al., 1998
).
Antisense and sense tfr1a and tfr1b probes were synthesized
from the respective cDNA clones in pGEM-T easy vector. Adult peripheral blood
and kidney tissue samples were isolated and Wright-Giemsa stained as described
(Ransom et al., 1996
;
Brownlie et al., 1998
).
Meiotic mapping
Genetic mapping strains were created by mating cia AB or Tü
heterozygotes to the polymorphic Dar or WIK strains. Embryos were collected
from pairwise matings of mapping strain cia heterozygotes, and scored
at 72 hours post fertilization (hpf) for hypochromia. Genomic DNA extraction
from individual embryos and bulk segregant analysis were performed as
described (Zhang et al., 1998)
using primers designed to SSLP markers obtained from the Massachusetts General
Hospital Zebrafish Server website
(http://zebrafish.mgh.harvard.edu)
and synthesized by Invitrogen.
Isolation of zebrafish Tfr genes, radiation hybrid mapping and mutation analysis
A 329 bp fragment of zebrafish tfr1a was isolated from zebrafish
kidney cDNA library using degenerate primers,
5'-TACACMCCWGGMTTCCC-3' (forward) and
5'-CCTGGRCCCCATGCATCCCTCTG-3' (reverse). This fragment was used to
screen zebrafish kidney cDNA gridded filters, which obtained partial clones of
tfr1b. For each tfr1, a combination of 5' and 3'
rapid amplification of cDNA ends (RACE) was used to determine the entire cDNA
sequence; full-length clones were then obtained by RT-PCR from 36 hpf embryos,
using 5'-ATGGATCAAGCCAGGACAACC-3' (forward) and
5'-CTAAAGAGGTGAGCTGAAG-3' (reverse) primers for tfr1a,
and 5'-ATGGCAGGAACAATTGGTCAA-3' (forward) and
5'-CTAGATTTCGTTGTCCAGGGA-3' (reverse) for tfr1b. A
partial fragment of tfr2 was cloned using online genome sequence
data, and the open reading frame determined by 5' RACE; a full-length
clone was obtained by RT-PCR from 36 hpf embryos using
5'-ATGATGGACTCGGTCACAGGA-3' (forward) and
5'-CTACAGCGGGTTGTCGATGTT-3' (reverse). Radiation hybrid mapping
was done with the following forward and reverse PCR primers: tfr1a,
5'-CAACAACATCCTCGTTCAG-3' and
5'-CTCTGGACCCCGATCACC-3'; tfr1b,
5'-GCTTCGACATCGACCAGGTGC-3' and
5'-GCACCTTGAAATGGGAGC-3'; and tfr2,
5'-CCCATCAGCAGATGAACCAACGAA-3' and
5'-ACATAGGTGTGTTTACCGTTTTCC-3'. Mutation analysis was done by
isolating cDNA from each cia allele at 36 hpf. tfr1a was
amplified using the primers above, subcloned into pGEM-T Easy vector
(Promega), and clones sequenced to determine the mutations.
cDNA overexpression constructs and morpholino designs
Full-length tfr1a and tfr1b cDNAs were subcloned into the
pCS2+ vector and mRNA synthesized using SP6 mMessage mMachine (Ambion).
mtfr1 and mtfr2 cDNA clones were a gift from Vera Sellers
(Children's Hospital, Boston), and were subcloned into pCS2+. For expression
in zebrafish ciaiu089 embryos, approximately 500-600 pg of
synthetic mRNA encoding tfr1a, tfr1b, mtfr1 or mtfr2 was
injected into 1-4-cell stage embryos.
Two morpholino antisense oligonucleotides targeting the tfr1a transcript were obtained from Gene-Tools: tfr1a-MO1 (5'-AGATGGTTGTCCTGGCTTGATCCAT-3') was designed to the predicted start codon (underlined); tfr1a-MO2 (5'-ACACCTTCGAGTGGACGAAGTAACAC-3') was designed to the splice donor of exon 13. Embryos were injected with 1 nl of either tfr1a-MO1 at 0.1 mg/ml or with tfr1a-MO2 at 1.5 mg/ml; to rescue tfr1a-MO1, embryos were co-injected with 500 pg of tfr1a cRNA. Morpholinos designed against the tfr1b transcript were as follows: tfr1b-MO1 (5'-CCAATTGTTCCTGCCATGGGATCTG-3') was designed against the predicted start codon (underlined), tfr1b-MO2 (5'-AACAAAACTTACCATTCTGGAAAC-3') and tfr1b-MO3 (5'-GCGGCTGTTTACCTATTAACAGAGG-3') were designed against the respective splice donor and acceptor sites between exons 1 and 2. Embryos were injected with 1 nl of tfr1b-MO1 at 1.25 mg/ml or co-injected with tfr1b-MO1/MO2 at 0.5 mg/ml each; to rescue tfr1b-MO1, embryos were co-injected with 300 pg of tfr1b mRNA.
Iron-dextran microinjection assays
Intravenous iron injection at 48 hpf was performed as previously described
(100 mg/ml, Sigma), such that each embryo received approximately 100 ng
iron-dextran (Donovan et al.,
2000). For 1-cell injections, iron-dextran was diluted to 10
mg/ml, and embryos injected with approximately 10 ng iron-dextran.
GenBank accession numbers
Zebrafish tfr1a, AY649363; zebrafish tfr1b, AY649364; and
zebrafish tfr2, AY649365.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
To determine whether mutations in tfr1a were present in the
various cia alleles, RT-PCR was used to obtain full-length cDNA
clones of tfr1a from embryos of each allele. Each cia allele
was found to harbor a mutation in either the tfr1a open reading frame
or a conserved splice site (Fig.
3B). Sequence analysis of tfr1a in
ciahp327 identified a T-to-A transversion at nucleotide
+1889 that results in an IN missense mutation at codon 630.
ciahs019 were found to have a G-to-A transition at
nucleotide +1970 that causes a G
D missense mutation at codon 657. The
residues mutated in ciahp327 and
ciahs019 are both located in the Tfr1a regions of the
helical domain involved in Tf binding
(Buchegger et al., 1996
;
Cheng et al., 2004
;
Lawrence et al., 1999
).
Mutagenesis studies have further localized the Tf/Tfr binding interface to
include a conserved RGD sequence, and mutation of the glycine in particular
severely abrogates Tf binding (Dubljevic
et al., 1999
; Giannetti et
al., 2003
; Liu et al.,
2003
; West et al.,
2001
). As the ciahs019 mutation occurs at this
particular glycine, we predict it may directly eliminate Tf binding, although
this tripeptide in Tfr1a is replaced by QGS residues. The residue mutated in
ciahp327 is located in
helix 1 of the helical
domain, adjacent to a lysine residue critical for Tf binding, and may
similarly disrupt interaction with the ligand
(Giannetti et al., 2003
;
Liu et al., 2003
). We found
ciatu25f possessed a G-to-A nucleotide transition at the
exon 13 splice donor site that results in inclusion of a 90 bp intron with a
premature stop codon, which eliminates the entire Tfr1 helical domain as well
as 94 residues (approximately 30%) of the protease domain. RT-PCR of
tfr1a from ciatu25f detected the presence of both
the wild-type tfr1a cDNA and the mis-spliced variant (data not
shown). This was consistent with the ciatu25f hypomorphic
phenotype, as it suggests that to some extent, ciatu25f
are capable of synthesizing normal Tfr1a. Lastly, we found
ciaiu089 had a T-to-C transition at nucleotide +946, with
a resulting F
L mis-sense mutation at codon 316. The F316 residue is
located on an apical domain loop that is altered in conformation upon Tfr
ligand binding, and although a mutation in this loop may cause its local
destabilization, it is not clear what the precise consequence might be for
overall Tfr function.
We next ascertained the expression pattern of each zebrafish tfr1 during embryogenesis. Whole-mount in-situ hybridization of zebrafish embryos showed tfr1a to be highly expressed in the developing blood, as marked by ße1 globin expression (Fig. 4A-H). tfr1a expression was first detected at 12 hpf in the ventral mesoderm, which converges to form the hematopoietic intermediate cell mass, the zebrafish intraembryonic blood island (data not shown). Blood-specific expression of tfr1a was maintained until 36 hpf in circulating primitive erythrocytes. By contrast, tfr1b was found to be expressed ubiquitously throughout embryogenesis (Fig. 4C,F,I). Notably, expression was not elevated above the level of ubiquitous expression in either developing or circulating primitive erythrocytes.
|
|
|
|
Zebrafish tfr1b is used for iron uptake by non-hematopoietic tissues
Based on the in-situ hybridization, as well as RT-PCR analyses from embryos
between 24 hpf and 6 dpf, it was evident that tfr1b was expressed
throughout embryogenesis (Fig.
4 and data not shown). To investigate the role of tfr1b
during development, we used antisense MOs to evaluate loss of tfr1b
function. In particular, we were interested in whether abrogation of Tfr1b
would affect hemoglobinization during erythroid cell differentiation. A MO
designed against the tfr1b translational start site (MO1) was
injected into wild-type zebrafish embryos at the 1-cell stage and the embryos
were examined throughout development. At 18-24 hpf, tfr1b
MO1-injected animals exhibited brain necrosis and growth delay. By 36-48 hpf,
embryos were still developmentally delayed, and could be grouped into three
classes according to their overall growth progression and the severity of
their nervous system necrosis (Table
2, Fig. 7). Class I
injected animals (7.8%) exhibited a moderate growth delay in comparison with
uninjected wild-type embryos (Fig.
7A-D). Class II injected embryos (87.5%) displayed severe growth
retardation, being much smaller with a markedly curved trunk and tail
(Fig. 7E,F). Class III injected
embryos (4.7%) included the morphants with extreme growth retardation
(Fig. 7G,H). All Class II and
III affected morphants died before 5 dpf. Despite this, all tfr1b
morphant groups underwent normal hemoglobinization, with visibly red blood in
circulation and o-dianisidine concentrations indistinguishable from
uninjected wild-type siblings. Similar results were observed with co-injection
of MOs targeted to the splice donor and acceptor sites at the junction between
exons 1 and 2 (data not shown).
|
|
Multiple Tfr genes are sufficient to rescue hypochromia in cia
We next determined if tfr1b could compensate for the loss of
tfr1a function in cia erythrocytes. We overexpressed
tfr1b in ciaiu089 mutants and found that
hemoglobin production was partially rescued in 27% of animals injected
(Fig. 5F;
Table 1). The number of
ciaiu089 rescued and the degree of rescue were similar to
that observed when tfr1a was overexpressed. These results confirm
that Tfr1b functions to deliver cellular iron. We also examined expression of
tfr1b in cia mutants to assess if its expression might be
altered, and hence partly accountable for the homozygous viability of
ciaiu089 and ciatu25f. However, we
detected no changes in tfr1b expression in any cia allele by
whole-mount in-situ hybridization (data not shown). Thus, taken together with
the morpholino data, this shows that while tfr1b is capable of iron
delivery into erythrocytes, it is not normally utilized by developing
erythroid cells.
As Tf receptors are conserved in structure throughout vertebrate evolution,
we wondered if multiple family members would be able to rescue cia
when similarly overexpressed in the embryo. In support of a common biochemical
function, we found that the overexpression of mouse tfr1 mRNA
partially rescued hemoglobin synthesis in 17% of ciaiu089
embryos (Fig. 5G,
Table 1). Again, the number of
cia embryos rescued and quantity of cells per embryo in which
hemoglobin was detected were similar to the overexpression experiments
conducted with tfr1a and tfr1b. Although the function of
Tfr2 in body iron metabolism has yet to be elucidated, mammalian Tfr2 is
capable of binding and transporting Tf-bound iron in vitro
(Kawabata et al., 1999;
Kawabata et al., 2001
;
West et al., 2000
). Based on
these data, we tested if overexpression of Tfr2 would rescue hemoglobin
production in cia. Injection of ciaiu089 mutants
with mouse tfr2 mRNA at the 1-cell stage partially rescued
hypochromia in 27% of mutants (Fig.
5H; Table 1). Thus
mammalian Tfr2 was also able to compensate for loss of tfr1a function
in erythroid cells. This series of Tfr overexpression experiments suggests
that the presence of any number of known Tf receptors on a differentiating
erythrocyte can facilitate iron uptake adequate for the production of
hemoglobin.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Functional equivalence among vertebrate Tfr genes in vivo
The overexpression of several Tfr genes in cia embryos was shown
to attenuate the cia hypochromic phenotype. These results illustrate
that when expressed in differentiating erythroid cells, a number of Tf
receptors are capable of mediating iron uptake sufficient for hemoglobin
biosynthesis. Our data specifically show the functional equivalence between
zebrafish tfr1a, zebrafish tfr1b, mouse Tfr1, and
mouse Tfr2 in the developing zebrafish. It is particularly noteworthy
that cia could be rescued with mouse Tfr2, as this is the first
in-vivo illustration of Tfr2 facilitating erythroid iron uptake. This finding
emphasizes that functional differences between mammalian Tfr1 and Tfr2 result
from differences in the spatiotemporal expression of these respective
genes.
Complementary roles of the zebrafish tfr1 duplicate genes during embryogenesis
The phenomenon of an erythroid-specific transferrin receptor in zebrafish
is most likely explained by a teleost genome duplication event postulated to
have happened following the split between teleost and tetrapod lineages
(Amores et al., 1998;
Gates et al., 1999
;
Postlethwait et al., 1998
;
Woods et al., 2000
). Based on
the presence of gene duplicates discovered in multiple teleosts, including
zebrafish, pufferfish and medaka, the genome duplication is predicted to have
occurred at least 100 million years ago in a teleost ancestor that pre-dates
the radiation of teleosts (Amores et al.,
1998
; Aparicio et al.,
1997
; Christoffels et al.,
2004
; Gates et al.,
1999
; Naruse et al.,
2000
; Santini and Tyler,
1999
; Taylor et al.,
2003
; Wittbrodt et al.,
1998
). Our finding that the summation of zebrafish Tfr1a and Tfr1b
functions equate to that of mammalian Tfr1 is consistent with the
subfunctionalization model of gene duplicate preservation, in which gene
copies retain part of the original gene's function
(Force et al., 1999
;
Ohno, 1970
;
Prince and Pickett, 2002
;
Zhang, 2003
). Common with
other examples of subfunctionalization, the respective expression patterns of
the tfr1a and tfr1b have diverged, such that together they
recreate the expression of the single ancestral gene, even though in this case
the proteins have interchangeable biochemical functions
(Bruce et al., 2001
;
Chiang et al., 2001
;
de Martino et al., 2000
;
Dorsky et al., 2003
;
Lister et al., 2001
;
Nornes et al., 1998
;
Oates et al., 1999
;
Pfeffer et al., 1998
). We
speculate that non-overlapping tfr1a and tfr1b expression
was made possible by the evolution of different sets of regulatory elements
for each duplicate. Future work in defining the promoter elements of
tfr1a and tfr1b will better characterize the potential
differences in the regulation and developmental expression of these genes.
Zebrafish as a vertebrate model to study the metabolism of iron and other essential metals
As part of our assessment of tfr1a function in erythropoiesis, we
developed a means to distinguish between cell extrinsic and intrinsic defects
in iron utilization. We utilized a previously characterized method of
zebrafish intravenous iron-dextran injection to determine if low plasma iron
was a factor in the failure of cia erythrocytes to hemoglobinize
normally (Donovan et al.,
2000). In this assay, the ability to rescue hemoglobin synthesis
with intravenously-supplied iron demonstrates that the erythrocytes are fully
capable of iron uptake, intracellular trafficking and metabolism; in direct
contrast, the inability to rescue indicates that the presence of a defect(s)
intrinsic to the mechanism of cellular iron uptake or utilization in
erythrocytes is present. Our de-novo method of iron-dextran injection at the
1-cell stage then serves to test whether developing erythrocytes can traffic
and metabolize intracellular iron subsequent to iron uptake. With this assay,
we believe that provision of excess iron to the embryo cytoplasm before the
onset of cleavage acts to bypass the later necessity for erythroid iron
internalization, because all cells in the embryo have been saturated with an
excess of usable iron. This novel combination of iron-dextran injection assays
is a valuable tool that can now be employed to categorize hypochromic mutants
with unknown gene defects currently being studied in our laboratory.
We found it surprising that injection of iron-dextran at the 1-cell stage was relatively non-toxic to the embryo, and we expect this forecasts broader applicability of similar assays. Single cell injection of any number of conjugated trace metals, such as copper or zinc, could be utilized to investigate their function and metabolism in a developmental setting. As we have done, the method could be applied to track various maternal yolk storage components, and could be applied to characterize the defect(s) in genetic mutants. Zebrafish present a unique opportunity to better understand the transit and utilization of iron and other metals during embryogenesis, and such studies in genetic mutants will enable investigation of the pathophysiology of numerous disease states.
In recent years, elucidation of the defects in several zebrafish with hypochromic anemia, in conjunction with the ongoing development of assays to understand their biology, have made significant contributions to the understanding of vertebrate iron metabolism. In this report we have presented evidence that cia represents a specific defect in erythrocyte iron uptake due to an ancestral duplication of the teleost tfr1 locus. Thus cia provide a model to further define the role of Tfr1 in erythropoiesis without a panorama of complicating tissue defects. Furthermore, the zebrafish system provides the ability to implement targeted genetic and chemical screens that could identify additional pathways with a role in the maintenance of iron homeostasis.
Tübingen 2000 Screen Consortium
F. Bebber van, E. Busch-Nentwich, R. Dahm, H. G. Frohnhöfer, H.
Geiger, D. Gilmour, S. Holley, J. Hooge, D. Jülich, H. Knaut, F.
Maderspacher, C. Neumann, T. Nicolson, C. Nüsslein-Volhard, H. Roehl, U.
Schönberger, C. Seiler, C. Söllner, M. Sonawane, A. Wehner, C.
Weiler and B. Schmidt at the Max-Planck-Institut für
Entwicklungsbiologie, Spemannstrasse 35, 72076 Tübingen, Germany.
U. Hagner, E. Hennen, C. Kaps, A. Kirchner, T. I. Koblizek, U. Langheinrich, C. Metzger, R. Nordin, M. Pezzuti, K. Schlombs, J. deSantana-Stamm, T. Trowe, G. Vacun, A. Walker and C. Weiler at Artemis Pharmaceuticals/Exelixis Deutchland GmbH, Neurather Ring 1, S51063 Köln, Germany.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
A list of the members of the Consortium and their affiliations is provided
at the end of the manuscript
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aisen, P. (2004). Molecules in focus: Transferrin receptor 1. Int. J. Biochem. Cell Biol. 36,2137 -2143.[CrossRef][Medline]
Amores, A., Force, A., Yan, Y. L., Joly, L., Amemiya, C., Fritz,
A., Ho, R. H., Langeland, J., Prince, V., Wang, Y. L. et al.
(1998). Zebrafish hox clusters and vertebrate genome evolution.
Science 282,1711
-1714.
Andrews, N. C. (1999). Disorders of iron
metabolism. N. Engl. J. Med.
341,1986
-1995.
Andrews, N. C. (2000). Iron homeostasis: insights from genetics and animal models. Nat. Rev. Genet. 1,208 -217.[CrossRef][Medline]
Aparicio, S., Hawker, K., Cottage, A., Mikawa, Y., Zuo, L., Venkatesh, B., Chen, E., Krumlauf, R. and Brenner, S. (1997). Organization of the Fugu rubripes Hox clusters: evidence for continuing evolution of vertebrate Hox complexes. Nat. Genet. 16,79 -83.[Medline]
Baker, E., Baker, S. M. and Morgan, E. M. (1998). Characterization of non-transferrin-bound iron (ferric citrate) uptake by rat hepatocytes in culture. Biochim. Biophys. Acta 1380,21 -30.[Medline]
Brownlie, A. and Zon, L. I. (1999). The zebrafish as a model system for the study of hematopoiesis. Bioscience 49,382 -392.
Brownlie, A., Donovan, A., Pratt, S. J., Paw, B. H., Oates, A. C., Brugnara, C., Witkowska, H. E., Sassa, S. and Zon, L. I. (1998). Postional cloning of the zebrafish sauternes gene: a model for congenital sideroblastic anemia. Nat. Genet. 20,244 -250.[CrossRef][Medline]
Brownlie, A., Hersey, C., Oates, A. C., Paw, B. H., Falick, A. M., Witkowska, H. E., Flint, J., Higgs, D., Jessen, J., Bahary, N. et al. (2003). Characterization of embryonic globin genes of the zebrafish. Dev. Biol. 255, 48-61.[CrossRef][Medline]
Bruce, A. E., Oates, A. C., Prince, V. E. and Ho, R. K. (2001). Additional hox clusters in the zebrafish: divergent expression patterns belie equivalent activities of duplicate hoxB5 genes. Evol. Dev. 3,127 -144.[CrossRef][Medline]
Buchegger, F., Trowbridge, I. S., Liu, L. F. S., White, S. and Collawn, J. F. (1996). Functional analysis of human/chicken transferrin receptor chimeras indicates that the carboxy-terminal region is important for ligand binding. Eur. J. Biochem. 235,9 -17.[Abstract]
Chan, R. Y., Ponka, P. and Schulman, H. M. (1992). Transferrin-receptor-independent but iron-dependent proliferation of variant Chinese hamster ovary cells. Exp. Cell Res. 202,326 -336.[Medline]
Cheng, Y., Zak, O., Aisen, P., Harrison, S. C. and Walz, T. (2004). Structure of the human transferrin receptor-transferrin complex. Cell 116,565 -576.[CrossRef][Medline]
Chiang, E. F. L., Pai, C. I., Wyatt, M., Yan, Y. L., Postlethwait, J. and Chung, B. C. (2001). Two sox9 genes on duplicated zebrafish chromosomes: expression of similar transcription activators in distinct sites. Dev. Biol. 231,149 -163.[CrossRef][Medline]
Christoffels, A., Koh, E. G., Chia, J. M., Brenner, S., Aparicio, S. and Venkatesh, B. (2004). Fugu genome analysis provides evidence for a whole-genome duplication early during the evolution of ray-finned fishes. Mol. Biol. Evol. Mar 10 Epub.
deMartino, S., Yan, Y. L., Jowett, T., Postlethwait, J. H., Varga, Z. M., Ashworth, A. and Austin, C. A. (2000). Expression of sox11 gene duplicates in zebrafish suggests the reciprocal loss of ancestral gene expression patterns in development. Dev. Dyn. 217,279 -292.[CrossRef][Medline]
Donovan, A., Brownlie, A., Zhou, Y., Shepard, J., Pratt, S. J., Moynihan, J., Paw, B. H., Drejer, A., Barut, B., Zapata, A. et al. (2000). Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter. Nature 403,776 -781.[CrossRef][Medline]
Donovan, A., Brownlie, A., Dorschner, M. O., Zhou, Y., Pratt, S.
J., Paw, B. H., Phillips, R. B., Thisse, C., Thisse, B. and Zon, L.
I. (2002). The zebrafish mutant gene chardonnay (cdy) encodes
divalent metal transporter 1 (DMT1). Blood
100,4655
-4659.
Dorsky, R. I., Itoh, M., Moon, R. T. and Chitnis, A.
(2003). Two tcf3 genes cooperate to pattern the zebrafish brain.
Development 130,1937
-1947.
Dubljevic, V., Sali, A. and Goding, J. W. (1999). A conserved RGD (Arg-Gly-Asp) motif in the transferrin receptor is required for binding to transferrin. Biochem. J. 341,11 -14.[CrossRef][Medline]
Fleming, R. E., Migas, M. C., Holden, C. C., Waheed, A.,
Britton, R. S., Tomatsu, S., Bacon, B. R. and Sly, W. S.
(2000). Transferrin receptor 2: continued expression in mouse
liver in the face of iron overload and in hereditary hemochromatosis.
Proc. Natl. Acad. Sci. USA
97,2214
-2219.
Fleming, R. E., Ahmann, J. R., Migas, M. C., Waheed, A.,
Koeffler, H. P., Kawabata, H., Britton, R. S., Bacon, B. R. and Sly, W.
S. (2002). Targeted mutagenesis of the murine transferrin
receptor-2 gene produces hemochromatosis. Proc. Natl. Acad. Sci.
USA 99,10653
-10658.
Force, A., Lynch, M., Pickett, F. B., Amores, A., Yan, Y. L. and
Postlewait, J. (1999). Preservation of duplicate genes
by complementary, degenerative mutations. Genetics
151,1531
-1545.
Gates, M. A., Kim, L., Egan, E. S., Cardozo, T., Sirotkin, H.
I., Dougan, S. T., Lashkari, D., Abagyan, R., Schier, A. F. and Talbot,
W. S. (1999). A genetic linkage map for zebrafish:
comparative analysis and localization of genes and expressed structures.
Genome Res. 9,334
-347.
Gelvan, D., Fibach, E., Meyron-Holtz, E. G. and Konijn, A.
M. (1996). Ferritin uptake by human erythroid precursors is a
regulated iron uptake pathway. Blood
88,3200
-3207.
Giannetti, A. M., Snow, P. M., Zak, O. and Bjorkman, P. J. (2003). Mechanism for multiple ligand recognition by the human transferrin receptor. PLoS Biol. 1, 341-350.[CrossRef]
Goto, Y., Paterson, M. and Listowski, I.
(1983). Iron uptake and regulation of ferritin synthesis by
heptoma cells in hormone-supplemented serum-free media. J. Biol.
Chem. 258,5248
-5255.
Haffter, P., Granato, M., Brand, M., Mullins, M. C.,
Hammerschmidt, M., Kane, D. A., Odenthal, J., van Eeden, F. J., Jiang,
Y. J., Heisenberg, C. P. et al. (1996). The identification of
genes with unique and essential functions in the development of the zebrafish,
Danio rerio. Development
123, 1-36.
Hentze, M. W., Muckenthaler, M. U. and Andrews, N. C. (2004). Balancing acts: molecular control of mammalian iron metabolism. Cell 117,285 -297.[CrossRef][Medline]
Hodgson, L. L., Quail, E. A. and Morgan, E. H. (1995). Iron transport mechanisms in reticulocytes and mature erythrocytes. J. Cell Physiol. 162,181 -190.[Medline]
Inman, R. S. and Wessling-Resnick, M. (1993).
Characterization of transferrin-independent iron transport in K562 cells.
J. Biol. Chem. 268,8521
-8528.
Kaplan, J., Jordan, I. and Sturrock, A. (1991).
Regulation of the transferrin-independent iron transport system in cultured
cells. J. Biol. Chem.
266,2997
-3004.
Kawabata, H., Yang, R., Hirama, T., Vuong, P. T., Kawano, S.,
Gombart, A. F. and Koeffler, H. P. (1999). Molecular
cloning of transferrin receptor 2. J. Biol. Chem.
274,20826
-20832.
Kawabata, H., Nakimaki, T., Ikonomi, P., Smith, R. D., Germain,
R. S. and Koeffler, H. P. (2001). Expression of transferrin
receptor 2 in normal and neoplastic hematopoietic cells.
Blood 98,2714
-2719.
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullman, B. and Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203,253 -310.[Medline]
Konijn, A. M., Meyron-Holtz, E. G., Fibach, E. and Gelvan, D. (1994). Cellular ferritin uptake: a highly regulated pathway for iron assimilation in human erythroid precursor cells. Adv. Exp. Med. Biol. 356,189 -197.[Medline]
Lawrence, C. M., Ray, S., Babyonyshev, M., Galluser, R.,
Borhani, D. W. and Harrison, S. C. (1999). Crystal structure
of the ectodomain of human transferrin receptor.
Science 286,779
-782.
Leimberg, J. M., Konijn, A. M. and Fibach, E. (2003). Developing human erythroid cells grown in transferrin-free medium utilize iron originating from extracellular ferritin. Am. J. Hematol. 73,211 -212.[CrossRef][Medline]
Levy, J. E., Jin, O., Fujiwara, Y., Kuo, F. and Andrews, N. C. (1999). Transferrin receptor is necessary for development of erythrocytes and the nervous system. Nat. Genet. 21,396 -399.[CrossRef][Medline]
Lister, J. A., Close, J. and Raible, D. W. (2001). Duplicate mitf genes in zebrafish: complementary expression and conservation of melanogenic potential. Dev. Biol. 237,333 -344.[CrossRef][Medline]
Liu, R., Guan, J. Q., Zak, O., Aisen, P. and Chance, M. R. (2003). Structural reorganization of the transferrin c-lobe and transferrin receptor upon complex formation: the c-lobe binds to the receptor helical domain. Biochemistry 42,12447 -12454.[CrossRef][Medline]
Meyron-Holtz, E. G., Vaisman, B., Cabantchik, Z. I., Fibach, E.,
Rouault, T. A., Hershko, C. and Konijn, A. M. (1999).
Regulation of intracellular iron metabolism in human erythroid precursors by
internalized extracellular ferritin. Blood
94,3205
-3211.
Naruse, K., Fukamachi, S., Mitani, H., Kondo, M., Matsuoka, T.,
Kondo, S., Hanamura, N., Morita, Y., Hasegawa, K., Nishigaki, R. et
al. (2000). A detailed map of medaka, oryzias latipes.
Comparative genomics and genome evolution. Genetics
154,1773
-1784.
Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene `knockdown' in zebrafish. Nat. Genet. 26,216 -220.[CrossRef][Medline]
Ned, R. M., Swat, W. and Andrews, N. C. (2003).
Transferrin receptor 1 is differentially required in lymphocyte development.
Blood 102,3711
-3718.
Nornes, S., Clarkson, M., Mikkola, I., Pederson, M., Bardsley, A., Martinez, J. P., Krauss, S. and Johansen, T. (1998). Zebrafish contains two Pax6 genes involved in eye development. Mech. Dev. 77,185 -196.[CrossRef][Medline]
Oates, A. C., Brownlie, A., Pratt, S. J., Irvine, D. V., Liao,
E. C., Paw, B. H., Dorian, K. J., Johnson, S. L., Postlethwait, J. H.,
Zon, L. I. et al. (1999). Gene duplication of zebrafish JAK2
homologs is accompanied by divergent embryonic expression patterns: only jak2a
is expressed during erythropoiesis. Blood
94,2622
-2636.
Ohno, S. (1970). Evolution by Gene Duplication. Heidelberg, Germany: Springer-Verlag.
Pfeffer, P. L., Gerster, T., Lun, K., Brand, M. and Busslinger,
M. (1998). Characterization of three novel members of the
zebrafish Pax2/5/8 family: dependency of Pax5 and Pax8 expression on the
Pax2.1 (noi) function. Development
125,3063
-3074.
Ponka, P. and Lok, C. N. (1999). The transferrin receptor: role in health and disease. Int. J. Biochem. Cell. Biol. 31,1111 -1137.[CrossRef][Medline]
Postlethwait, J. H., Yan, Y. L., Gates, M. A., Horne, S., Amores, A., Brownlie, A., Donovan, A., Egan, E. S., Force, A., Gong, Z. et al. (1998). Vertebrate genome evolution and the zebrafish gene map. Nat. Genet. 18,345 -349.[Medline]
Postlethwait, J. H., Woods, I. G., Ngo-Hazelett, P., Yan, Y. L.,
Kelly, P. D., Chu, F., Huang, H., Hill-Force, A. and Talbot, W. S.
(2000). Zebrafish comparative genomics and the origins of
vertebrate chromosomes. Genome Res.
10,1890
-1902.
Prince, V. E. and Pickett, F. B. (2002). Splitting pairs: the diverging fates of duplicated genes. Nat. Rev. Genet. 3,827 -837.[CrossRef][Medline]
Ransom, D. G., Haffter, P., Odenthal, J., Brownlie, A.,
Vogelsang, E., Kelsh, R. N., Brand, M., van Eeden, F. J. M.,
Furutani-Seiki, M., Granato, M. et al. (1996).
Characterization of zebrafish mutants with defects in embryonic hematopoiesis.
Development 123,311
-319.
Santini, F. and Tyler, J. C. (1999). A new phylogenetic hypothesis for the order Tetraodontiformes (Teleostei, Pisces), with placement of the most fossil basal lineages. Am. Zool. 39,10A .
Sturrock, A., Alexander, J., Lamb, J., Craven, C. M. and Kaplan,
J. (1990). Characterization of a transferrin-independent
uptake system for iron in HeLa cells. J. Biol. Chem.
265,3139
-3145.
Taylor, J. S., Braasch, I., Frickey, T., Meyer, A. and Van de
Peer, Y. (2003). Genome duplication, a trait shared by 22,000
species of ray-finned fish. Genome Res.
13,382
-390.
Thompson, M. A., Ransom, D. G., Pratt, S. J., MacLennan, H., Kieran, M. W., Detrich, H. W., 3rd, Vial, B., Huber, T. L., Paw, B., Brownlie, A. J., Oates, A. C., Fritz, A., Gates, M. A., Amores, A., Bahary, N., Talbot, W. S., Her, H., Beier, D. R., Postlethwait, J. H. and Zon, L. I. (1998). The cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis. Dev. Biol. 197,248 -269.[CrossRef][Medline]
Thorstensen, K., Trinder, D., Zak, O. and Aisen, P. (1995). Uptake of iron from N-terminal half-transferrin by isolated rat hepatocytes. Evidence of transferrin-receptor-independent iron uptake. Eur. J. Biochem. 232,129 -133.[Abstract]
Trinder, D. and Baker, E. (2003). Transferrin receptor 2: a new molecule in iron metabolism. Int. J. Biochem. Cell Biol. 35,292 -296.[CrossRef][Medline]
West, A. P., Bennett, M. J., Sellers, V. M., Andrews, N. C.,
Enns, C. A. and Bjorkman, P. J. (2000). Comparison of
the interactions of transferrin receptor and transferrin receptor 2 with
transferrin and the hereditary hemochromatosis protein HFE. J.
Biol. Chem. 275,38135
-38138.
West, A. P., Giannetti, A. M., Herr, A. B., Bennett, M. J., Nangiana, J. S., Pierce, J. R., Weiner, L. P., Snow, P. M. and Bjorkman, P. J. (2001). Mutational analysis of the transferrin receptor reveals overlapping HFE and transferrin binding sites. J. Mol. Biol. 313,385 -397.[CrossRef][Medline]
Westerfield, M. (1993). The Zebrafish Book. Eugene, Oregon: University of Oregon Press.
Wingert, R. A. and Zon, L. I. (2003). Genetic dissection of hematopoiesis using the zebrafish. In Hematopoietic Stem Cells (ed. I. Godin and A. Cumano), pp.1 -18. Georgetown: Texas: Landes Bioscience.
Wittbrodt, J., Meyer, A. and Schartl, M. (1998). More genes in fish? BioEssays 20,511 -515.[CrossRef]
Woods, I. G., Kelly, P. D., Chu, F., Ngo-Hazelett, P., Yan, Y.
L., Huang, H., Postlethwait, P. H. and Talbot, W. S.
(2000). A comparative map of the zebrafish genome.
Genome Res. 10,1903
-1914.
Yang, J., Goetz, D., Li, J. Y., Wang, W., Mori, K., Setlik, D., Du, T., Erdjuent-Bromage, H., Tempst, P., Strong, R. et al. (2002). An iron delivery pathway mediated by a lipocalin. Mol. Cell 10,1045 -1056.[Medline]
Zhang, J. (2003). Evolution by gene duplication: an update. Trends Ecol. Evol. 18,292 -298.[CrossRef]
Zhang, J., Talbot, W. S. and Schier, A. F. (1998). Positional cloning identifies zebrafish one-eyed pinhead as a permissive EGF-related ligand required during gastrulation. Cell 92,241 -251.[Medline]
Related articles in Development: