Blood, 15 May 2002, Vol. 99, No. 10, pp. 3873-3875
CORRESPONDENCE
To the editor:
Mouse surviving solely on human erythropoietin receptor (EpoR):
model of human EpoR-linked disease
Mouse models of human diseases have contributed immensely to our
understanding of the function of genes and their mutations and to the
disease phenotype. In some cases, the compatibility of human and mouse
signal transduction interactions allows replacement of the mouse genes
with their human counterparts. In those cases, disease-causing
mutations from patients can be introduced into the mouse germ line
allowing direct testing of the gene defect in vivo. Different genetic
approaches have been used to create animal models of human diseases
caused by gain-of-function mutations. Human transgenes have been bred
onto its mouse homologue knock-out background,1 or the
human gene has been used to directly replace its murine homologue (ie,
knocked-in into the mouse gene locus).2 A necessary
assumption of a functional mutant human gene in the mouse environment
is the full compatibility of these genes in both species.
Using the transgenic approach, Yu et al3 have recently
shown that the human erythropoietin receptor (EpoR) can rescue
erythropoiesis and all other developmental defects associated with the
mouse EpoR deficiency. The 80-kb human EpoR transgene recapitulated EpoR expression not only in hematopoietic tissues but also in other
tissues known to express EpoR. The human EpoR-rescued mice exhibited
normal hematologic parameters (including hematocrit) and normal numbers
of erythroid progenitors in the bone marrow. However, Yu et
al3 also concluded that mouse erythropoietin (Epo) and
human EpoR are fully compatible in vivo and questioned earlier
observations of others that murine Epo has reduced activity on human
cells.4
We think that this conclusion is not warranted and provide the
following evidence. We replaced the mouse EpoR gene with its human
wild-type and mutant homologue in murine embryonic stem (ES)
cells.2 Animals homozygous for the human wild-type human EpoR gene were slightly, but significantly, anemic compared to their
littermates (average hematocrit, 45% vs 49%). Additionally, the
animals homozygous for the human wild-type EpoR had lower levels of
early erythroid progenitors in the bone marrow (in average 12 vs 19 erythroid colonies per 105 cells plated as assessed in
semisolid media containing Epo) and smaller spleens before and after
phenylhydrazine injection. These data are either compatible with a
lower efficiency of in vivo interaction of mouse Epo with human EpoR as
suggested by Nicolini et al4 or may be explained by a
possible lower efficiency of interaction of the human EpoR with the
mouse downstream signaling molecules. Nevertheless, these data prove
that mouse and human Epo/EpoR signaling are functionally compatible but
have quantitative differences in signaling molecule interactions.
The mutant human EpoR that we knocked-in into the mouse EpoR locus was
cloned from a patient with primary familial and congenital polycythemia
(PFCP).5 We have shown that mice heterozygous or
homozygous for the truncated gain-of-function human EpoR were polycythemic and their erythroid progenitors had increased in vitro
sensitivity to both mouse and human Epo.2 Interestingly, another animal model of truncated EpoR without apparent polycythemic phenotype in adults and with comparable in vitro responses of wild-type
and mutant erythroid progenitors to Epo was later published by Zang et
al.6 The reason for this discrepancy is not clear; in both
cases the resulting truncated EpoR contained one tyrosine. However, it
is possible that the mutant EpoR isolated from the subject with PFCP
has different functional properties than the artificially truncated
mouse EpoR used by Zang et al.6 The levels of expression
of the mutant EpoR were comparable to the wild-type mouse EpoR in both
studies; however, in our model the level of expression was
achieved only after removal of the neomycin (neo)-selectable marker by crossing the mice
to cre-transgenic mice (Figure
1). The animals reported by Zang et
al6 had the neo gene retained in the EpoR locus,
albeit driven by a different promoter than that used by us.
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| Figure 1.
Expression of the human EpoR in the mouse EpoR locus.
Primer extension of reverse transcriptase-polymerase chain reaction
(RT-PCR) product was used to evaluate relative levels of mouse and
human EpoR mRNAs, as described.2 The genotypes of ES cells
(first lane) or mice (all other lanes) are indicated. The ES cells in
the first lane were in vitro-differentiated into embryoid bodies in
semisolid media with Epo.7 The cultures were harvested at
day 9 of differentiation, and the cells were used as a template for
RT-PCR. The presence of a neo-selectable marker did not
suppress human EpoR gene expression in the mouse EpoR locus in vitro.
In the other lanes, bone marrow cells were used as a template for
RT-PCR. The retention of a neo cassette in the human EpoR
gene generated a null or hypomorphic allele in vivo. The presence (+ neo) or absence ( neo) of this selectable
marker gene is displayed. wt indicates wild type; mt,
mutant.
|
|
While these data indicate that the differences between mouse and human
cellular environment exist, they do not undermine the usefulness of the
mouse models of human diseases. Our objective, a comparison of disabled
and reactivated gain-of-function Epo/EpoR signaling in different
tissues through cre-mediated recombination, should help our
understanding of the role of Epo signaling in nonerythroid tissues and
elucidate the increased risk of cardiovascular disease in PFCP.
Vladimir Divoky and Josef T. Prchal
Correspondence: Josef Prchal, Baylor College of Medicine, One
Baylor Plaza, Suite 802E, Houston, TX 77030; e-mail:
jprchal{at}bcm.tmc.edu
References
1.
Ryan TM, Ciavatta DJ, Townes TM.
Knockout-transgenic mouse model of sickle cell disease.
Science.
1997;278:873-876[Abstract/Free Full Text].
2.
Divoky V, Liu Z, Ryan TM, Prchal JF, Townes TM, Prchal JT.
Mouse model of congenital polycythemia: homologous replacement of murine gene by mutant human erythropoietin receptor gene.
Proc Natl Acad Sci U S A.
2001;98:986-991[Abstract/Free Full Text].
3.
Yu X, Lin CS, Costantini F, Noguchi CT.
The human erythropoietin receptor gene rescues erythropoiesis and developmental defects in the erythropoietin receptor null mouse.
Blood.
2001;98:475-477[Abstract/Free Full Text].
4.
Nicolini FE, Holyoake TL, Cashman JD, Chu PP, Lambie K, Eaves CJ.
Unique differentiation programs of human fetal liver stem cells shown both in vitro and in vivo in NOD/SCID mice.
Blood.
1999;94:2686-2695[Abstract/Free Full Text].
5.
Prchal JT.
Pathogenetic mechanisms of polycythemia vera and congenital polycythemic disorders.
Semin Hematol.
2001;38:10-20[Medline]
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6.
Zang H, Sato K, Nakajima H, McKay C, Ney PA, Ihle JN.
The distal region and receptor tyrosines of the Epo receptor are non-essential for in vivo erythropoiesis.
EMBO J.
2001;20:3156-3166[CrossRef][Medline]
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Helgason CD, Sauvageau G, Lawrence HJ, Largman C, Humphries RK.
Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro.
Blood.
1996;87:2740-2749[Abstract/Free Full Text].
Response:
Equivalence of mouse and human erythropoietin/erythropoietin
receptor signaling
Divoky et al have produced mice expressing the full-length and
truncated human EPOR that exhibit mild anemia and
polycythemia respectively.1 Based on comparisons with mice
expressing endogenous erythropoietin receptor (Epor) or a
truncated mouse Epor,2 they suggest that mouse
and human EPO/EPOR signaling are compatible but are
different quantitatively in their interactions. Such differences would
have important implications for mouse models of human diseases based on
human EPOR expression and signaling.
An explanation for differences in mice expressing human EPOR
may not reflect different signaling interactions between human and
mouse EPO/EPOR but rather the levels of EPOR
expression. We found that a human EPOR transgene (80 kb)
with expression comparable to the endogenous mouse Epor at
all phases of development (yolk sac, fetal liver, and adult spleen and
bone marrow)3 quantitatively rescues the mouse
Epor null phenotype.4 Hemoglobin, hematopoietic progenitors (erythroid burst-forming unit, granulocyte macrophage colony-forming unit, granulocyte-erythrocyte-megakaryocyte-macrophage colony-forming unit), and response to anemic stress (reticulocyte count
and hematocrit) are similar to control mice. No differences in spleen
size were noted, and all animals exhibit an increase in spleen size
upon phenylhydrazine treatment. Although higher levels of
Epo induction were detected in the
mEpor
/ hEPOR+ mice
after phenylhydrazine treatment (2.5 that of control animals), hematologic parameters suggest that expression of this human
EPOR transgene behaves largely analogous to endogenous mouse
Epor. This transgene also corrects the increased apoptosis
associated with mEpor/ fetal brain and heart
development.5
The construction of the human EPOR gene by Divoky et
al1 differ markedly from our human EPOR
transgene. We previously carried out careful analysis of the human
EPOR proximal region and found that while much of the
transcription activity is contained in a 15-base pair (bp) 5' proximal
promoter fragment, contributions can be seen from the more distal
flanking regions.6 This is also observed in reporter gene
experiments in transgenic mice for promoter fragments extending to
150-bp 5' and 1778-bp 5' (Z. Y. Liu, C. Liu, and C. T. Noguchi, unpublished data, 2002). Additional flanking
sequences appear to be required to provide a high level of human
EPOR expression in vivo. For the intact human
EPOR transgene, we made more than 15 transgenic lines with
low copy number using 3 different genomic fragments. The construct
extending only 2 kb 5' provides an appropriate but low level of
tissue-specific EPOR expression, about an order of magnitude
lower than the endogenous mEpor.7 A
significantly higher level of expression on the order of the endogenous
gene is observed with 2 other constructs containing longer 5' regions.
Both of these human EPOR gene fragments are able to rescue
the mEpor/ phenotype, and the 80-kb human
EPOR fragment was chosen for further study.3
This transgene shows appropriate expression in a variety of tissues
quantitatively comparable to endogenous mouse Epor, particularly in hematopoietic tissue.
Both the mouse and human proximal promoters contain requisite GATA-1
and SP1 binding sites and are homologous in this
region.6,8 However, homology of the 5' flanking region
does not extend much beyond 
60 bp 5'.9 To target the
mouse locus, Divoky et al used a human EPOR gene fragment
(DraI) extending to 550 bp 5' fused to the 5' flanking
mouse Epor region at 300 bp
(NaeI).1 This construct retains a
species-specific upstream repetitive element that inhibits
transcription of the mouse Epor,10 and the 550 bp proximal promoter fragment for human EpoR is considerably less than the 1778 bp fragment that resulted in the low level of
transgene expression in vivo.7 Based on our studies of
human EPOR gene expression, the differences in the 5' region
flanking the human EPOR genes used to produce the 2 mouse
models are significant. Variations in human EPOR expression
due to differences in background strain or site of gene integration are
also possible. Careful quantification of EPOR during
development and in adult mice would clarify if a lower level of human
EPOR expression in mice produced by Divoky et al accounts
for the associated mild anemia without invoking differences between
mouse and human EPO/EPOR signaling.
As another piece of evidence that mouse and human EPO/EPOR
signaling are different, Divoky and Prchal point to the production of
mice expressing truncated Epor with only a single
cytoplasmic tyrosine at 343.1,2 After initial reports of a
negative regulatory domain in the cytoplasmic tail of mouse
Epor11 and human
EPOR,12 other truncated deletions in human
EPOR have been associated with polycythemia including the
mutation constructed by Divoky et al.1 These mice express
human EpoR with a truncated deletion just before tyrosine
410 and exhibit a polycythemia phenotype as predicted from the human
condition. Interestingly, in addition to direct alteration of
EPO signaling, increased cell-surface expression via
posttranslational mechanism(s) has been suggested for a truncated EPOR associated primary familial and congenital
polycythemia.13 Other mice expressing a truncated mouse
EpoR were constructed by altering the endogenous
Epor sequence 3' of the HindIII site resulting in
a truncated mouse Epor with a single cytoplasmic tyrosine
that is about 2 dozen amino acids shorter than the Divoky and Prchal
deletion.2 These mice exhibit largely constitutive erythropoiesis and are not polycythemic. While it is possible that
these differences may be inherent in mouse versus human
EPOR, variation in erythrocytosis may also result from the
extent or level of expression of the truncations.
Recapitulation of the human phenotype with the truncated human
EPOR by Divoky et al1 confirms the similarity of
EPO/EPOR signaling in mouse and human and the potential for
using mouse models to understand EPO/EPOR interactions. The
differences noted here among the EPOR mouse models
illustrate the advantages and limitations of manipulating the mouse
genome to alter phenotypic expression. When comparing model systems,
careful analysis of gene construction in vivo, mouse strains, and
possible integration sites are necessary to ensure comparable
expression and analogous gene products. Note that while the original
targeting construct for human EPOR by Divoky and Prchal was
active in vitro, it was necessary to remove the neomycin
(neo)-selectable marker from human EPOR intron 6 to obtain expression in vivo, although the neo gene remains
3' flanking the endogenous mouse Epor produced by Zang et
al.1,2 Variation in gene expression can become significant, particularly when the differences are small. While human
and mouse EPO/EPOR signaling may not be equivalent, we do not see any evidence that they are significantly different in our
EPO/EPOR animal model.
Xiaobing Yu and Constance Tom Noguchi
Correspondence: Constance Tom Noguchi, Laboratory of Chemical
Biology, National Institute of Diabetes and Digestive and Kidney
Diseases, National Institutes of Health, 10 Center Dr MSC-1822,
Bethesda, MD 20892-1822
References
1.
Divoky V, Liu Z, Ryan TM, Prchal JF, Townes TM, Prchal JT.
Mouse model of congenital polycythemia: homologous replacement of murine gene by mutant human erythropoietin receptor gene.
Proc Natl Acad Sci U S A.
2001;98:986-991.
2.
Zang H, Sato K, Nakajima H, McKay C, Ney PA, Ihle JN.
The distal region and receptor tyrosines of the Epo receptor are non-essential for in vivo erythropoiesis.
EMBO J.
2001;20:3156-3166.
3.
Liu C, Shen K, Liu Z, Noguchi CT.
Regulated human erythropoietin receptor expression in mouse brain.
J Biol Chem.
1997;272:32395-32400[Abstract/Free Full Text].
4.
Yu X, Lin CS, Costantini F, Noguchi CT.
The human erythropoietin receptor gene rescues erythropoiesis and developmental defects in the erythropoietin receptor null mouse.
Blood.
2001;98:475-477.
5.
Yu X, Shacka JJ, Eells JB, et al.
Erythropoietin receptor signalling is required for normal brain development.
Development.
2002;129:505-516[Abstract/Free Full Text].
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Chin K, Oda N, Shen K, Noguchi CT.
Regulation of transcription of the human erythropoietin receptor gene by proteins binding to GATA-1 and Sp1 motifs.
Nucleic Acids Res.
1995;23:3041-3049[Abstract/Free Full Text].
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Liu ZY, Chin K, Noguchi CT.
Tissue specific expression of human erythropoietin receptor in transgenic mice.
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Zon LI, Youssoufian H, Mather C, Lodish HF, Orkin SH.
Activation of the erythropoietin receptor promoter by transcription factor GATA-1.
Proc Natl Acad Sci U S A.
1991;88:10638-10641[Abstract/Free Full Text].
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Noguchi CT, Bae KS, Chin K, Wada Y, Schechter AN, Hankins WD.
Cloning of the human erythropoietin receptor gene.
Blood.
1991;78:2548-2556[Abstract/Free Full Text].
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Youssoufian H, Lodish HF.
Transcriptional inhibition of the murine erythropoietin receptor gene by an upstream repetitive element.
Mol Cell Biol.
1993;13:98-104[Abstract/Free Full Text].
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D'Andrea AD, Yoshimura A, Youssoufian H, Zon LI, Koo JW, Lodish HF.
The cytoplasmic region of the erythropoietin receptor contains nonoverlapping positive and negative growth-regulatory domains.
Mol Cell Biol.
1991;11:1980-1987[Abstract/Free Full Text].
12.
de la Chapelle A, Traskelin AL, Juvonen E.
Truncated erythropoietin receptor causes dominantly inherited benign human erythrocytosis.
Proc Natl Acad Sci U S A.
1993;90:4495-4499[Abstract/Free Full Text].
13.
Motohashi T, Nakamura Y, Osawa M, et al.
Increased cell surface expression of C-terminal truncated erythropoietin receptors in polycythemia.
Eur J Haematol.
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