Suppressor screen in Mpl?/?mice: c-Myb mutation
causes supraphysiological production of platelets in
the absence of thrombopoietin signaling
Marina R. Carpinelli*†, Douglas J. Hilton*†, Donald Metcalf*, Jennifer L. Antonchuk*, Craig D. Hyland*,
Sandra L. Mifsud*, Ladina Di Rago*, Adrienne A. Hilton*, Tracy A. Willson*, Andrew W. Roberts*, Robert G. Ramsay‡,
Nicos A. Nicola*, and Warren S. Alexander*§
*The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville VIC 3052, Australia; and‡The Peter MacCallum Cancer Centre,
St. Andrews Place, East Melbourne VIC 3002, Australia
Contributed by Donald Metcalf, March 2, 2004
Genetic screens in lower organisms, particularly those that identify
modifiers of preexisting genetic defects, have been used success-
fully to order components of complex signaling pathways. To date,
similar suppressor screens have not been used in vertebrates. To
define the molecular pathways regulating platelet production, we
have executed a large-scale modifier screen with genetically
thrombocytopenic Mpl?/?mice by using N-ethyl-N-nitrosourea
mutagenesis. Here we show that mutations in the c-Myb gene
cause a myeloproliferative syndrome and supraphysiological ex-
pansion of megakaryocyte and platelet production in the absence
of thrombopoietin signaling. This screen demonstrates the utility
screens in mice for the simultaneous discovery and in vivo valida-
tion of targets for therapeutic discovery in diseases for which
mouse models are available.
tion at the genomic level that mutations in humans can often
recapitulate disease when introduced into the germline of mice.
germline mutagen in mice, genetic screens have now become
feasible in a mammalian model of direct relevance to human
health and disease (1). Some ENU mutagenesis screens in mice
have focused on particular traits such as circadian rhythm or on
particular regions of the genome, whereas others have screened
for anomalies in a wide range of tissues and organs (2–6). Unlike
screens in lower organisms (7–9), to date all of these studies have
begun with wild-type mice and have isolated mutants with
abnormal traits. The successful application of modifier screens
in lower organisms, with prominent examples including the
dissection of sevenless-dependent eye development in Drosoph-
ila melanogaster (10) and vulval development in Caenorhabditis
elegans (11), led us to explore the use of suppressor screens in
In this study, we describe a large-scale suppressor screen, the
aim of which was to identify mutations capable of ameliorating
thrombocytopenia, a lack of blood platelets. Blood platelets are
shed by megakaryocytes into the circulation, where they are
required for blood clotting and hemostasis. Thrombopoietin
(TPO), acting through its specific cell surface receptor c-Mpl, is
considered the principal cytokine controlling megakaryocyte
and platelet numbers (12, 13). Mice and humans lacking func-
tional Tpo or Mpl genes are profoundly thrombocytopenic and
have a corresponding reduction in the numbers of megakaryo-
cytes, megakaryocyte progenitor cells, and stem cells (14–21).
We screened 1,575 Mpl?/?mice harboring random ENU-
induced mutations for amelioration of thrombocytopenia, re-
sulting in isolation of two independent partial loss-of-function
alleles of c-Myb. When homozygous, these mutations produced
a supraphysiological expansion of megakaryocyte and platelet
ice and humans are physiologically similar, are afflicted by
many of the same diseases, and show sufficient conserva-
production in the absence of signaling by the major regulator of
Materials and Methods
Generation and Screening of Mutant Mice. Male Mpl?/?C57BL?6
mice (14) were treated with a total dose of 200–400 mg?kg ENU
divided into one, two, or three weekly injections, as described
(22). Four weeks after the final injection, ENU-treated mice
were mated with one or two isogenic female mice to yield
first-generation (G1) progeny. At 7 weeks of age, blood from G1
mice was collected from the retroorbital plexus into tubes
containing potassium EDTA (Sarstedt), and the number of
platelets in the peripheral blood was determined by using an
Advia 120 automated hematological analyzer (Bayer, Tarry-
Test for Linkage Between Plt3 and Plt4. To determine whether Plt3
the progeny were bled at 7 weeks of age to determine their
platelet count. These mice were then bred with ??? Mpl?/?
mice, and the numbers of platelets in these progeny were
determined at 7 weeks of age.
Analyses of Epistasis. Plt4?Plt4 Mpl?/?mice were mated to ???
Mpl?/?mice to produce Plt4/? Mpl?/?animals, which were then
intercrossed. The Mpl genotype of the progeny was determined
by Southern blot (14), and the genotype of the Plt4 locus was
inferred from one or two generation progeny testing. The
phenotypes of Plt4?Plt4 Mpl?/?, Plt4?Plt4 Mpl?/?, and ???
Mpl?/?mice were then compared to assess the epistatic rela-
tionship of the genes.
Genetic Mapping. Affected heterozygous Plt4/? Mpl?/?mice on
a C57BL?6 background were crossed to Mpl?/?mice on a 129Sv
background. Plt4/? Mpl?/?F1animals were then identified at 7
weeks of age because of their elevated platelet counts and
intercrossed to produce 65 mice in the F2generation. At 7 weeks
of age, F2mice were bled and their peripheral blood platelet
numbers were determined. Mice were killed, and DNA was
prepared from a piece of liver according to described methods
(23). One hundred forty-eight simple sequence length polymor-
phisms (SSLPs) spaced evenly throughout the genome were
amplified and analyzed, essentially as described (24). Plt4 was
found to reside on chromosome 10, and its location was refined
through analysis of additional markers in the region. Tight
Abbreviations: ENU, N-ethyl-N-nitrosourea; TPO, thrombopoietin.
†M.R.C. and D.J.H. contributed equally to this work.
§To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2004 by The National Academy of Sciences of the USA
April 27, 2004 ?
vol. 101 ?
no. 17 ?
genetic linkage of Plt3 to Plt4 was confirmed by crossing Plt3??
Mpl?/?mice on a C57BL?6 background to ??? Mpl?/?mice on
a 129Sv background. Plt3/? Mpl?/?F1 animals were then
identified at 7 weeks of age because of their elevated platelet
counts and were backcrossed to ??? Mpl?/?mice on a 129Sv
background to generate N2 mice. The platelet counts of 68
Mpl?/?N2mice were determined at 7 weeks of age, and their
genotype at SSLP markers closely linked to Plt4 was assessed.
DNA Sequencing. Mice generated by intercrossing either Plt3/?
Mpl?/?or Plt4/? Mpl?/?mice were typed as Plt3?Plt3, Plt3/?,
Plt4?Plt4, Plt4/?, or ??? at 7 weeks of age by determining their
weeks of age, DNA was prepared, and each of the exons of the
c-Myb gene (25) was amplified by PCR and sequenced on an
Applied Biosystems automatic sequencer according to the man-
Transactivation Assays. To compare the activity of c-MybPlt3and
c-MybPlt4with that of wild type c-Myb and a constitutively active
truncation mutant (CT3) of c-Myb (26), the respective cDNAs
were cloned into the pEF-BOS vector (27). The c-Myb expres-
sion constructs were then cotransfected into 293 cells with a
reporter construct containing five consecutive high-affinity c-
the CAT gene (a gift of Joe Lipsick, Stanford University,
Stanford, CA), and transactivation activity was measured as
described (28). The expression of the various c-Myb alleles was
measured by Western blotting by using the Mab1.1 and Mab 5.1
antibodies as described (29).
Hematological Analysis. The hematocrit, platelet and white cell
count, and differential were determined by using either manual
or automated (Advia 120, Bayer) counting techniques. Clonal
cultures of hemopoietic cells were performed as described (23).
Cultures of 2.5 ? 104adult bone marrow cells or 5 ? 104spleen
cells in 1 ml of 0.3% agar in DMEM supplemented with newborn
calf serum (20%) were stimulated with a mixture of 100 ng?ml
murine stem cell factor, 10 ng?ml murine IL-3, and 4 units?ml
human EPO and incubated for 7 days at 37°C in a fully
humidified atmosphere of 10% CO2in air. Agar cultures were
fixed; sequentially stained for acetylcholinesterase, Luxol fast
blue, and hematoxylin; and the cellular composition of each
colony determined at ?100–400. Megakaryocyte counts were
performed by manual counting from sections of sternum and
spleen after staining with hematoxylin?eosin. A minimum of 10
high-power fields (?200) were scored. Colony-forming units
(spleen) were enumerated by i.v. injection of bone marrow cells
into recipient mice that had been irradiated with 11 Gy of
?-irradiation in two equal doses given 3 hours apart from a137Cs
source (Atomic Energy, Ottawa). Transplanted mice were main-
tained on oral antibiotic (1.1 g?liter neomycin sulfate; Sigma).
Spleens were removed after 12 days, fixed in Carnoy’s solution
(60% ethanol?30% chloroform?10% acetic acid), and the num-
bers of macroscopic colonies were counted.
Flow Cytometry. Single-cell suspensions of spleen and bone
marrow cells were depleted of erythrocytes by lysis with 156 mM
ammonium chloride (pH 7.3). Cells were stained with a satu-
rating concentration of IgM-FITC and B220–phycoerythrin
(PE) or Ter119-PE and CD71-FITC (30) (BD Pharmingen), and
analyses were performed on an LSR flow cytometer (BD
Biosciences, San Diego). Dead cells were excluded based on
propidium iodide staining.
A Large-Scale Random Mutagenesis Screen to Isolate Suppressors of
Thrombocytopenia. The aim of this genetic screen was to identify
mutations capable of ameliorating thrombocytopenia. The mice
we used had markedly reduced platelet numbers (117 ? 52 ?
106?ml, n ? 783 vs. 1,168 ? 230 ? 106platelets per ml, n ? 100
for isogenic C57BL?6 controls, Fig 1 a and b), caused by a
loss-of-function deletion in the c-Mpl gene, which encodes the
cell surface receptor for the platelet-regulating cytokine,
Three hundred Mpl?/?C57BL?6 mice were injected with
200–400 mg?kg ENU in one, two, or three weekly doses. Four
weeks after injection, males were mated with isogenic Mpl?/?
C57BL?6 female mice to produce first-generation (G1) off-
spring. At 7 weeks of age, we analyzed the peripheral blood of
1,575 G1 Mpl?/?mice and found five mice with ?300 ? 106
platelet?ml, a count ?3 SD higher than the mean observed in
Mpl?/?C57BL?6 mice (Fig. 1 c, d, and g).
To determine whether suppression of thrombocytopenia was
heritable, the five G1animals were crossed to untreated Mpl?/?
mice, and the platelet numbers in their progeny were measured.
The progeny of two of the five mice (named Plt3 and Plt4)
contained individuals with the suppressed phenotype, and this
trait showed simple Mendelian inheritance consistent with a
dominant phenotype (Fig. 1 e and h). In one case, the G1mouse
died before yielding progeny, whereas in the remaining two
cases, suppression was not heritable (data not shown). Because
each of the affected G1 mice was derived from a different
ENU-treated Mpl?/?mouse, this suggested that independent
dominant suppressors of thrombocytopenia had been isolated.
Plt3 and Plt4 Cause Semidominant Phenotypes with Homozygous Mice
Exhibiting Supraphysiological Increases in Platelet Numbers. Al-
though heterozygous Plt3 and Plt4 mutations led to amelioration
of thrombocytopenia, platelet counts did not reach wild-type
levels. We wished to determine whether platelet counts were
further elevated in homozygous mice. Offspring from intercross-
ing Plt4?? Mpl?/?mice displayed either the low platelet counts
typical of Mpl?/?mice, mild amelioration of thrombocytopenia,
or supraphysiological platelet levels (Fig. 1i). These phenotypes
were shown to correspond to ??? Mpl?/?, Plt4/? Mpl?/?, and
Plt4?Plt4 Mpl?/?genotypes by breeding experiments. Notably,
when Plt4?Plt4 Mpl?/?mice were crossed to ??? Mpl?/?mice,
all of the progeny had mild suppression of thrombocytopenia
characteristic of Plt4/? Mpl?/?mice; whereas if Plt4?Plt4 Mpl?/?
mice were intercrossed, then all of the pups exhibited supra-
physiological platelet numbers characteristic of their parents
(Fig. 1 j and k). Although the proportions of these three
phenotypes were consistent with full penetrance of the pheno-
type and viability of the homozygotes for Plt4, a lower than
expected frequency of homozygotes was observed for Plt3 (Fig.
1f), and this was shown to be due to embryonic or neonatal death
of a proportion of the homozygous mice rather than a lack of
penetrance of the phenotype (data not shown).
Plt3 and Plt4 Are Independent Hypomorphic Alleles of c-Myb. To
determine whether Plt3 and Plt4 were genetically linked, Plt3-
Plt4 compound heterozygotes were produced and mated to
Mpl?/?mice. All progeny of the Plt3-Plt4 compound heterozy-
gotes exhibited mild amelioration of thrombocytopenia, typical
Plt3 and Plt4 mutations are tightly linked and could be alleles of
the same gene (data not shown).
To obtain a chromosomal localization for the closely linked
Plt3 and Plt4 mutations, Plt4/? Mpl?/?mice (C57BL?6 back-
ground) were mated with ??? Mpl???mice on a 129Sv back-
ground and then Plt4/? Mpl?/?(C57BL?6 ? 129Sv)F1progeny
were intercrossed to produce 65 F2 generation mice. Platelet
counts in these F2mice varied from the very low levels expected
of ??? Mpl?/?mice, intermediate levels characteristic of Plt4/?
Mpl?/?mice, and exceptionally high levels characteristic of
Plt4?Plt4 Mpl?/?mice. Using a set of 148 simple sequence length
www.pnas.org?cgi?doi?10.1073?pnas.0401496101Carpinelli et al.
polymorphisms, the only region of the genome in which linkage
was observed was at the centromeric end of chromosome 10
between markers D10Mit213 and D10Mit214 (Fig. 2a). Analysis
of 68 similarly generated backcross progeny demonstrated that
this interval was also closely linked to the Plt3 mutation (Fig. 2a).
Within this chromosomal region, the c-Myb locus represented
a compelling candidate for the location of the Plt3 and Plt4
mutations, because c-Myb mutations have been found to elevate
platelet numbers (31, 32). The coding regions of the entire c-Myb
gene from Plt3/?, Plt3?Plt3, Plt4/?, and Plt4?Plt4 Mpl?/?mice
were sequenced and compared with that from ??? Mpl?/?and
??? Mpl?/?mice. A single A to T transversion in the c-Myb
coding sequence was discovered in both mutants, resulting in
substitution of the valine for an aspartic acid codon at residue
152 of the c-Myb DNA-binding domain in the Plt3 allele and at
residue 384 in the leucine-zipper domain of the Plt4 allele (Fig.
2b). Northern blot analysis revealed that c-MybPlt3and c-MybPlt4
and, on transfection of expression vectors into 293 cells, the Plt3
and Plt4 c-Myb proteins were produced at levels similar to
wild-type c-Myb (data not shown). In contrast, compared to
wild-type c-Myb and a constitutively active c-Myb mutant, there
was a profound reduction in the activity of c-MybPlt4and a more
modest reduction of c-MybPlt3proteins in a transactivation assay
that partial loss-of-function of c-Myb can ameliorate thrombo-
cytopenia was confirmed by taking advantage of a previously
generated mouse (33) with deletion of one allele of the c-Myb
gene (c-Myb?/?) and showing that, similar to c-MybPlt3/?Mpl?/?
and c-MybPlt4/?Mpl?/?mice, c-Myb?/?Mpl?/?mice had signif-
icantly higher numbers of platelets than c-Myb?/?Mpl?/?mice
cytopoiesis. To investigate the biological basis for the ameliora-
tion of Mpl?/?thrombocytopenia due to loss of function of
c-Myb, we analyzed megakaryocytopoiesis in Plt3 and Plt4
cyte progenitor cells and megakaryocytes in the bone marrow
and spleen of heterozygous and, to a greater extent, homozygous
3a), consistent with the increase in platelet numbers in these
mice being the result of expanded cellular production within the
megakaryocyte lineage. The numbers of progenitor cells com-
mitted to other hemopoietic lineages were not significantly
altered in heterozygous Plt3 or Plt4 mutants; however, in ho-
mozygous Plt3 and Plt4 mice, the numbers of all progenitor cells
were elevated compared with control Mpl?/?mice (Table 1).
Perturbations in the multipotent progenitor compartment seem
likely to account for this observation, because significant in-
creases in the numbers of spleen colony-forming cells accom-
panied these changes (Fig. 3a). Although the numbers of spleen
colony-forming units in the bone marrow of homozygous Plt3
heterozygous or Mpl?/?cells.
Plt3 and Plt4 Homozygous Mice Exhibit Defects in Erythroid and B
Lymphoid Lineages. With the exception of megakaryocytopoiesis,
this multilineage myelodysplastic state did not result in excessive
production of other types of mature blood cells in homozygous
mutants (Table 1). The proportions of morphologically recog-
nizable cells in the granulocyte series were slightly elevated in
bone marrow and spleen of Plt3 and Plt4 mutants, but this was
likely to be at least in part an indirect effect of reduced lymphoid
cell production (see below), and monocyte numbers were rela-
tively unchanged (data not shown). Previous studies (31, 32)
have found that reduced expression of c-Myb results in defective
production of cells in the lymphoid and erythroid lineages. Our
analyses of the effects of reduced c-Myb function in Plt3 and Plt4
mutants supports these observations. Histological examination
of the spleen revealed that abnormally small lymphoid follicles
and expanded red pulp with atypical architecture accompanied
the greatly expanded numbers of megakaryocytes (Fig. 3c).
Significantly reduced numbers of B lymphoid cells were evident
in the bone marrow and spleen (Fig. 3b and data not shown), and
homozygous Plt3 and Plt4 mice were leukopenic, due largely to
reduced numbers of circulating lymphocytes (Table 1). Similarly,
to Mpl?/?mice (e), the progeny derived from crossing heterozygous Plt3/? Mpl?/?mice (f), the G1Plt4 mouse (g), the G2progeny derived from mating the G1
Plt4 mouse to Mpl?/?mice (h), the progeny derived from intercrossing heterozygous Plt4/? Mpl?/?mice (i), the progeny derived from mating homozygous
Plt4?Plt4 Mpl?/?mice to Mpl?/?mice (j), and the progeny derived from intercrossing homozygous Plt4?Plt4 Mpl?/?mice (k).
Identification of ENU-mutant mice with ameliorated thrombocytopenia. Peripheral blood platelet counts at 7 weeks of age for 100 Mpl?/?mice (a), 783
Carpinelli et al. PNAS ?
April 27, 2004 ?
vol. 101 ?
no. 17 ?
a large increase in the numbers of colony-forming unit erythroid
c-MybPlt3/Plt3, 155 ? 143; Mpl?/?c-MybPlt4/Plt4,154 ? 92; Mpl?/?
c-Myb?/?, 3 ? 3 per 105cells), defects in erythroid maturation
were evident in the bone marrow (Fig. 3b), and the animals were
mildly anemic (Table 1).
c-Myb Is Epistatic to Mpl. An advantage of modifier screens is their
capacity to order genes in a pathway using analyses of epistasis.
Accordingly, we assessed the phenotype of homozygous Plt4
mutants on a wild-type background (c-MybPlt4/Plt4Mpl?/?) in
comparison with that of wild-type (c-Myb?/?Mpl?/?) and
c-MybPlt4/Plt4Mpl?/?mice. Remarkably, the supraphysiological
production of platelets, megakaryocytes, and megakaryocyte
progenitors observed in c-MybPlt4/Plt4mice was independent of
the Mpl genotype (Table 1 and Fig. 3).
Previous studies have implied a complex role for c-Myb in
regulation of hemopoiesis (31–33). Deletion of the c-Myb gene
results in embryonic death due to failure of fetal liver hemo-
poiesis (33). In contrast, mice bearing a hypomorphic allele of
c-Myb survive and exhibit increased megakaryocyte production
but diminished production of erythroid and lymphoid lineages
(32). This spectrum of phenotypes is reproduced in mice with
Plt3 or Plt4 mutations consistent with partial loss of Myb
function. However, the availability of hypomorphic point muta-
tions in Myb should allow a finer dissection of the physiological
roles of specific c-Myb subdomains. In this regard, a similar
phenotype to that in Plt3?Plt3 and Plt4?Plt4 mice was observed
when a germline mutation in the KIX domain of p300, which
disrupts Myb binding, was established on a Myb?/?background
(31). These data suggest that disrupted interaction of Myb with
p300 predisposes to thrombocytosis. It will therefore be of
interest to determine whether the mutations that arose in Plt3
and Plt4 mice affect the interaction of Myb with p300. Moreover,
the data here establish, quite unexpectedly, that reduction of
c-Myb function results in a supraphysiological megakaryocyto-
poiesis and platelet production in the complete absence of
signaling by TPO.
The question arises whether inhibition of c-Myb and TPO
represents part of the same pathway or lies on independent
routes leading to platelet formation. To begin to address this
issue, we assessed the epistatic relationship of c-Myb and Mpl by
comparing the number of platelets observed in c-MybPlt4/Plt4
Mpl?/?mice with those of c-MybPlt4/Plt4Mpl?/?and c-Myb?/?
Mpl?/?mice. We observed a similar increase in platelet number
to between 3,000 and 4,000 ? 106?ml in c-MybPlt4/Plt4mice
whether c-Mpl was present or absent. One interpretation of this
result is that a critical step in the pathway by which TPO
generates megakaryocytes and platelets is down-regulation of
c-Myb expression or activity; hence when c-Myb activity is
reduced by mutation, the hemopoietic system of the animal
responds in a manner analogous to exposure to a high concen-
tration of TPO. Biochemical analyses of c-Myb expression and
action after TPO stimulation of hemopoietic progenitor cells
and megakaryocytes will be required to test this hypothesis.
Comprehensive genome-wide and targeted mutagenesis
screens using wild-type mice have been reported recently (2, 3,
5, 6). This paper describes a large-scale modifier screen in
vertebrates and demonstrates that the strategies that have
proven so valuable in yeast, worms, and flies (7–9) are also
applicable in higher organisms. In addition to their use in
dissecting complex biological processes, we propose that sup-
pressor screens in vertebrates are of potential value for the
identification of targets for drug discovery. Just as most ENU-
induced mutations cause loss of function, most small-molecule
therapeutics also reduce the function of proteins to which they
bind. Accordingly, screens for genes that, when mutated, lead to
amelioration of disease should provide genome-wide access to
novel in vivo validated targets for pharmaceutical discovery in
c-Myb. (a) To map Plt4, a (C57BL?6 ? 129Sv)F2generation of 65 mice was
produced, bled at 7 weeks of age, and categorized as having low platelets
(?150 ? 106?ml) characteristic of ??? Mpl?/?mice, moderate numbers of
extremely high platelets (?2,000 ? 106?ml) characteristic of Plt4?Plt4 Mpl?/?
mice. Animals were then genotyped, and markers found to be homozygous
129?Sv are shown in white, markers that were heterozygous are shown in
gray, and markers homozygous C57BL?6 are shown in black. The number of
to between D10Mit213 and D10Mit 214. To confirm Plt3 was located close to
Plt4, we produced 68 (C57BL?6 ? 129Sv)N2generation mice, measured plate-
let numbers in these animals, and genotyped them by using the markers most
closely linked to Plt4. (Right) Correlation between genotype and phenotype
for markers at the centromeric region of chromosome 10. (b) Sequence of
PCR-amplified exons and intron boundaries of the c-Myb gene showing a
of three Plt3?Pl3 Mpl?/?, three Plt3?? Mpl?/?mice, three Plt4?Plt4 Mpl?/?
mice, three Plt4?? Mpl?/?mice, three ??? Mpl?/?mice, and three ???
(CT3; ref. 26) were compared by measuring production of chloramphenicol
acetyltransferase from a c-Myb responsive promoter (28). The activity of both
Plt3 and Plt4 c-Myb was significantly lower than wild type; (P ? 0.017 and P ?
0.0003, respectively; n ? 9).
Plt3 and Plt4 are tightly linked on chromosome 10 and are alleles of
www.pnas.org?cgi?doi?10.1073?pnas.0401496101 Carpinelli et al.
Table 1. Hematological profile of Plt3 and Plt4 mutant mice
White cells (?10?3?ml)
Colony-forming progenitor cells
BM (per 2.5 ?104cells)
Spleen (per 5?104cells)
116 ? 52
52.7 ? 2.1
6.30 ? 1.57
0.48 ? 0.46
5.54 ? 1.16
0.39 ? 0.45
0.07 ? 0.10
4,662 ? 851
43.9 ? 2.3
3.26 ? 1.09
0.79 ? 0.35
2.06 ? 0.73
0.39 ? 0.20
0.02 ? 0.02
507 ? 132
50.1 ? 5.3
7.26 ? 2.7
0.89 ? 0.71
5.70 ? 2.56
0.38 ? 0.15
0.11 ? 0.07
4371 ? 718
43.3 ? 2.5
4.76 ? 1.41
1.60 ? 0.59
1.94 ? 0.85
0.89 ? 0.52
0.22 ? 0.28
449 ? 77
50.0 ? 5.9
6.67 ? 1.22
0.58 ? 0.61
4.92 ? 2.95
0.52 ? 0.42
0.15 ? 0.01
1,479 ? 128
51.3 ? 1.8
7.80 ? 1.94
0.43 ? 0.19
6.89 ? 1.86
0.36 ? 0.29
0.11 ? 0.08
3936 ? 618
45.3 ? 4.8
4.73 ? 0.35
1.53 ? 0.52
2.49 ? 0.21
0.60 ? 0.15
0.12 ? 0.09
4 ? 3
12 ? 4
9 ? 4
7 ? 3
0.9 ? 0.9
9 ? 6
17 ? 4
44 ? 15
19 ? 9
14 ? 12
0 ? 0
113 ? 16
4 ? 2
14 ? 4
11 ? 5
7 ? 3
1 ? 1
26 ? 6
21 ? 4
11 ? 5
31 ? 9
17 ? 5
0 ? 0
96 ? 24
10 ? 3
17 ? 6
11 ? 4
10 ? 5
0.7 ? 1.2
25 ? 14
11 ? 4
19 ? 4
12 ? 2
8 ? 5
3 ? 3
21 ? 6
12 ? 6
23 ? 20
27 ? 1
14 ? 1
0 ? 0
96 ? 25
0.1 ? 0.2
0.4 ? 0.5
0.5 ? 0.8
0.4 ? 0.8
0.1 ? 0.4
4 ? 5
5 ? 4
12 ? 5
7 ? 5
5 ? 4
0 ? 0
100 ? 23
1 ? 0.8
0 ? 0
0 ? 0
0.5 ? 0.1
0 ? 0
6 ? 5
3 ? 4
4 ? 3
3 ? 3
4 ? 7
0 ? 0
91 ? 105
0.2 ? 0.3
0 ? 0
0 ? 0
0.2 ? 0.3
0 ? 0
3 ? 2
1 ? 1
0.6 ? 0.8
0.2 ? 0.4
0.2 ? 0.4
0 ? 0
7 ? 7
2 ? 1.4
8 ? 1.4
12 ? 6
15 ? 8
0 ? 0
126 ? 24
Means ? SD of data from 15–50 (blood data) and 2–6 (progenitor cell data) mice of each genotype. BM, bone marrow.
units (spleen), a measure of multipotential progenitor cells, in the bone marrow (Far Left), clonogenic megakaryocyte progenitor cells (Left), megakaryocytes
background are shown. Assays were performed as described in Materials and Methods with the error bars representing the SD from the mean of data from 3–7
[colony-forming units (spleen)], 2–6 (progenitor cell data), 2–7 (megakaryocyte data), and 4–50 (platelet data) mice. (b) Flow cytometric analysis of B lymphoid
cells in the spleen and erythroid cells in the bone marrow of Plt4 mutant mice showing marked reductions in preB (B220?IgM?) and B (B220?IgM?) lymphocytes
from Mpl?/?c-Myb?/?, Mpl?/?c-MybPlt3/Plt3, Mpl?/?c-MybPlt4/Plt4, and Mpl?/?c-MybPlt4/Plt4mice. Note the poor development of lymphoid follicles and expanded
red pulp displaying reduced cellularity and disrupted architecture.
Mutation of c-Myb results in an elevation in progenitor cells, megakaryocytes, and platelets independent of Mpl. (a) The numbers of colony-forming
Carpinelli et al. PNAS ?
April 27, 2004 ?
vol. 101 ?
no. 17 ?
diseases with unmet clinical need for which mouse models are Download full-text
Jason Corbin, Naomi Sprigg, Janelle Mighall, Sally Cane, Elizabeth Viney,
Elaine Major, Kathy Hanzinikolas, Theresa Gibbs, Fiona Berryman, Ben
Radford, Chris Evans, Shauna Ross, Sonia Guzzardi, Jaclyn Cushen, Enza
Brullo, Tracey Kemp, and Amanda Hoskins. This work is supported by the
National Health and Medical Research Council, Canberra, Australia (Pro-
gram Grant 257500); the Anti-Cancer Council of Victoria, Melbourne,
Australia; the J. D. and L. Harris Trust; and MuriGen Pty Ltd.
1. Hitotsumachi, S., Carpenter, D. A. & Russell, W. L. (1985) Proc. Natl. Acad.
Sci. USA 82, 6619–6621.
2. Hrabe de Angelis, M. H., Flaswinkel, H., Fuchs, H., Rathkolb, B., Soewarto,
D., Marschall, S., Heffner, S., Pargent, W., Wuensch, K., Jung, M., et al. (2000)
Nat. Genet. 25, 444–447.
3. Kile, B. T., Hentges, K. E., Clark, A. T., Nakamura, H., Salinger, A. P., Liu,
B., Box, N., Stockton, D. W., Johnson, R. L., Behringer, R. R., et al. (2003)
Nature 425, 81–86.
4. King, D. P., Zhao, Y., Sangoram, A. M., Wilsbacher, L. D., Tanaka, M.,
Antoch, M. P., Steeves, T. D., Vitaterna, M. H., Kornhauser, J. M., Lowrey,
P. L., et al. (1997) Cell 89, 641–653.
5. Herron, B. J., Lu, W., Rao, C., Liu, S., Peters, H., Bronson, R. T., Justice, M. J.,
McDonald, J. D. & Beier, D. R. (2002) Nat. Genet. 30, 185–189.
6. Nolan, P. M., Peters, J., Strivens, M., Rogers, D., Hagan, J., Spurr, N., Gray,
I. C., Vizor, L., Brooker, D., Whitehill, E., et al. (2000) Nat. Genet. 25, 440–443.
7. Forsburg, S. L. (2001) Nat. Rev. Genet. 2, 659–668.
8. Jorgensen, E. M. & Mango, S. E. (2002) Nat. Rev. Genet. 3, 356–369.
9. St. Johnston, D. (2002) Nat. Rev. Genet. 3, 176–188.
10. Daga, A. & Banerjee, U. (1994) Cell Mol. Biol. Res. 40, 245–251.
11. Clark, S. G., Stern, M. J. & Horvitz, H. R. (1992) Nature 356, 340–344.
12. Kaushansky, K. (1995) Thromb. Haemostasis 74, 521–525.
13. Kaushanksy, K. & Drachman, J. G. (2002) Oncogene 21, 3359–3367.
14. Alexander, W. S., Robert, A. W., Nicola, N. A., Li, R. & Metcalf, D. (1996)
Blood 87, 2162–2170.
15. Alexander, W. S. (1999) Int. J. Biochem. Cell Biol. 31, 1027–1035.
16. De Sauvage, F., Carver-Moore, K., Luoh, S., Ryan, A., Dowd, M., Eaton, D. L.
& Moore, M. W. (1996) J. Exp. Med. 183, 651–656.
17. de Sauvage, F. J., Villeval, J. L. & Shivdasani, R. A. (1998) J. Lab. Clin. Med.
18. Gurney, A. L., Carver-Moore, K., de Sauvage, F. J. & Moore, M. W. (1994)
Science 265, 1445–1447.
19. Ghilardi, N., Wiestner, A., Kikuchi, M., Ohsaka, A. & Skoda, R. C. (1999) Br. J.
Haematol. 107, 310–316.
20. Ihara, K., Ishii, E., Eguchi, M., Takada, H., Suminoe, A., Good, R. A. & Hara,
T. (1999) Proc. Natl. Acad. Sci. USA 96, 3132–3136.
21. Kimura, S., Roberts, A. W., Metcald, D. & Alexander, W. S. (1998) Proc. Natl.
Acad. Sci. USA 95, 1195–1200.
22. Bode, V. C. (1984) Genetics 108, 457–470.
23. Alexander, W. S., Metcalf, D., Dunn, A. R. (1995) EMBO J. 14,
& Lander, E. S. (1992) Genetics 131, 423–447.
25. Klempnauer, K. H., Gonda, T. J. & Bishop, J. M. (1982) Cell 31, 453–463.
26. Ramsay, R. G., Ishii, S. & Gonda, T. J. (1991) Oncogene 6, 1875–1879.
27. Mizushime. (1990) Nucleic Acids Res. 18, 5322.
28. Chen, R. H. & Lipsick, J. S. (1993) Mol. Cell. Biol. 13, 4423–4431.
29. Ramsay, R. G., Ishii, S., Nishina, Y., Soe, G. & Gonda, T. J. (1989) Oncogene
Res. 4, 259–269.
30. Socolovsky, M., Fallon, E. E. J., Wang, S., Brugnara, C. & Lodish, H. F. (1999)
Cell 98, 181–191.
J. M. & Brindle, P. K. (2002) Nature 419, 738–743.
32. Emambokus, N., Vegiopoulos, A., Harman, B., Jenkinson, E., Anderson, G. &
Frampton, J. (2003) EMBO J. 22, 4478–4488.
33. Mucenski, M. L., McLain, K., Kier, A. B., Swerdlow, S. H., Schreiner, C. M.,
Miller, T. A., Pietryga, D. W., Scott, W. J., Jr., & Potter, S. S. (1991) Cell 65,
www.pnas.org?cgi?doi?10.1073?pnas.0401496101 Carpinelli et al.