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HEMATOPOIESIS AND STEM CELLS
A recessive screen for genes regulating hematopoietic stem cells
Peter Papathanasiou,1 Robert Tunningley,1 Diwakar R. Pattabiraman,2 Ping Ye,2 Thomas J. Gonda,2 Belinda Whittle,1
Adam E. Hamilton,1 Simon O. Cridland,3 Rohan Lourie,4 and Andrew C. Perkins3,4
1Australian Phenomics Facility, John Curtin School of Medical Research, Australian National University, Acton, Australia; 2Diamantina Institute for Cancer,
Immunology and Metabolic Medicine and 3Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia; and 4Department of Pathology,
Mater Hospital, Brisbane, Australia
Identification of genes that regulate the
development, self-renewal, and differentiation of stem cells is of vital importance for understanding normal organogenesis and cancer; such knowledge
also underpins regenerative medicine.
Here we demonstrate that chemical mutagenesis of mice combined with advances in hematopoietic stem cell reagents and genome resources can
efficiently recover recessive mutations
and identify genes essential for generation and proliferation of definitive hematopoietic stem cells and/or their progeny.
We used high-throughput fluorescenceactivated cell sorter to analyze 9 subsets
of blood stem cells, progenitor cells, circulating red cells, and platelets in more
than 1300 mouse embryos at embryonic
day (E) 14.5. From 45 pedigrees, we
recovered 6 strains with defects in definitive hematopoiesis. We demonstrate
rapid identification of a novel mutation
in the c-Myb transcription factor that
results in thrombocythemia and myelofibrosis as proof of principal of the
utility of our fluorescence-activated cell
sorter–based screen. Such phenotypedriven approaches will provide new
knowledge of the genes, protein interactions, and regulatory networks that underpin stem cell biology. (Blood. 2010;
116(26):5849-5858)
Introduction
Derived from the Greek word for blood (␣␫␮␣, haima), hematopoiesis describes the generation of fully differentiated effector blood
cells from multipotential, self-renewing hematopoietic stem cells
(HSCs).1 There are 2 waves of hematopoiesis in vertebrates (Figure
1A). The primitive wave is derived from hemangioblasts that form
in the posterior primitive streak and migrate onto the yolk sac (YS)
from embryonic day 7 (E7.0)2,3 where they give rise to primitive
erythroid progenitors (EryP) in association with endothelial and
vascular smooth muscle cells. EryP proliferate and differentiate
synchronously to generate large nucleated red cells, which enter the
circulation as the heart begins to beat at approximately E9.0.3,4
Platelets are also generated in the YS and enter the fetal circulation
early, where they may or may not perform a classic hemostatic
function.5 The definitive hematopoietic wave expands in the fetal
liver (FL) from approximately E11.5, but HSCs are produced
before this time in the ventral wall of the dorsal aorta and YS. Their
potential to make at least 8 different mature blood cell lineages is
not realized until the FL bud is formed from the gut tube and a
suitable microenvironment is established for HSC seeding. From
E11.5 to E15.5, there is a massive expansion of the FL, which is
initially geared for production of definitive enucleated red blood
cells to sustain rapid fetal growth (Figure 1A). From approximately
E15.5, myeloid lineages are produced and hepatocytes begin to
differentiate within the FL as it transforms from a hematopoietic
organ into an adult liver.
We know much about the genes that are critical for definitive
stem cell generation and differentiation from reverse genetic
approaches in mice, primarily gene knockouts.6 Most of the
essential transcription factors were initially discovered as onco-
genes, as proteins that bind to important cis-regulatory elements in
other genes, or by sequence homology.1,7 Some factors play dual
roles in primitive and definitive hematopoiesis, whereas others are
relatively definitive-specific. c-Myb is an example of the latter,8
whereas Scl/Tal-1 and Gata2 play essential roles in both waves.9,10
Forward genetic screens using the mutagen ethylnitrosourea
(ENU) offer several advantages over reverse genetics for gene
discovery.11 Most importantly, they are phenotype-driven and so
make no presumptions about which genes are involved in a
particular process. Resulting point mutations also mimic those that
characterize the majority of human inherited and acquired genetic
diseases, and point mutations can lead to hypomorphic or antimorphic alleles in addition to loss-of-function alleles. The ability of
mis-sense mutations to radically affect proteome regulatory networks and subsequent phenotypes has been well demonstrated in
both yeast12 and bacteria.13 Mammalian examples are also beginning to be described from ENU mouse screens,14-16 including at the
stem cell level.17 Gain-of-function (dominant mutations) and
precise mutations in critical cis-regulatory elements are also
possible outcomes from ENU screens. These are difficult to
achieve using reverse engineering technologies without leaving
small marks (such as LoxP sites) behind.
Several important genes for HSC and blood cell production
have been discovered through ENU in zebrafish.18 Initial screens
focused on embryonic anemia so were biased toward finding
mutations in patterning or primitive hematopoiesis. Whereas some
mutations were found in known hematopoietic genes,19 many more
were in unexpected genes.20,21 Our screen builds on numerous
successful dominant or haploinsufficient hematopoietic22,23 and
Submitted April 7, 2010; accepted June 20, 2010. Prepublished online as Blood
First Edition paper, July 7, 2010; DOI 10.1182/blood-2010-04-269951.
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 USC section 1734.
The online version of this article contains a data supplement.
© 2010 by The American Society of Hematology
BLOOD, 23 DECEMBER 2010 䡠 VOLUME 116, NUMBER 26
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BLOOD, 23 DECEMBER 2010 䡠 VOLUME 116, NUMBER 26
PAPATHANASIOU et al
(2) access a hematopoietic organ (FL) containing quantifiable
subsets of HSC/progenitors, (3) reduce time to screening of the
animal, and (4) reduce project costs. To date, we have screened
more than 1300 G3 embryos from 45 pedigrees for 9 hematopoietic
parameters in parallel and identified 6 recessive mutations. We
have given these pedigrees Australian Aboriginal names to reflect
the Australian origin of the screen, and as acknowledgment that our
Aboriginal peoples understand land sustainability just as stem cells
maintain and repair adult tissues after damage or degeneration.
Methods
Mice
All 8 wild-type strains, C57BL/6J (B6), C57BL/10SnJ (B10), C57L/J,
BALB/b, C3H/HeH, CBA/CaJ, FVB/NJ, and 129/SvEv, and ENU pedigrees (B6) were maintained at the Australian Phenomics Facility. The
morning of vaginal plug observation was E0.5. ENU pedigree founder (G0)
B6 male mice were treated weekly with 90 mg/kg ENU over 3 weeks. All
mouse experiments were approved by the Institutional Review Board of
Australian National University.
Flow cytometry
A full list of antibodies is available in supplemental data (available on the
Blood Web site; see the Supplemental Materials link at the top of the online
article). All cells were analyzed on an LSRII (BD Biosciences) and FACS
data analyzed using FlowJo Version 9.1 software (TreeStar).
Hematologic analyses
Whole blood (175 ␮L) from adult mice was collected into ethylenediaminetetraacetic acid tubes and run through an Advia 120/2120 Hematology
System analyzer (Siemens AG). Cytospins were at 500g for 4 minutes in a
Cytospin 3 (Shandon). Cells were fixed in 100% methanol and stained in
May-Grünwald-Giemsa. Paraffin sections of spleen were stained with
hematoxylin and eosin, Perl stain, or reticulin using standard protocols
(supplemental data).
Figure 1. Development of FACS-based embryonic screening assays.
(A) Schematic of hematopoietic development focusing on the circulating blood cell
types and FL progenitor cell types assayed in our E14.5 screen. Early blood
progenitors exit the posterior primitive streak (pps) and differentiate on the YS before
entering the circulation from when the heart begins to beat at approximately E9.0.
Large platelets also develop from the YS and enter the circulation early. Definitive
HSCs enter and exit the midprimitive streak (mps) slightly later and migrate to the
aorta-gonad-mesonephros (AGM) region where they develop with the ventral wall of
the dorsal aorta where they lie dormant until the FL buds provides a receptive niche
for HSC seeding and proliferation. In the FL, a hierarchy of progressively restricted
progenitors is produced. Early in ontogeny, MEPs provide a high output of enucleated
red cells and platelets. Later, myeloid progenitors generate neutrophils, and still later
lymphoid progenitors (CLPs) seed the bone marrow and thymus where they provide
B and T cells, respectively. We elected to screen the blood and FL at E14.5 so as to
gain access to primitive and definitive blood cells (⬃ 50:50 mix), and all definitive
progenitor cell types. (B) The FL screen assays 6 functionally distinct HSC/progenitor
cell subsets, LT-HSCs, MPPs, CLPs, CMPs, GMPs, and MEPs, by 8-color FACS
analysis. (C) The FB screen assays 3 distinct subsets of megakaryo/erythroid cells,
YS-derived primitive RBCs, FL-derived definitive RBCs, and platelets, by 3-color
FACS analysis and cell size (on logarithmic scale). A representative plot of tissues
from an E14.5 C57BL/6J embryo is shown, along with the percentage of nucleated
blood cells (B) and percentage of total blood (C).
immunologic11,14 screens to identify ENU phenodeviants in mice.
We describe 2 complementary fluorescence-activated cell sorter
(FACS)–based assays to detect perturbations in subsets of definitive HSC/progenitors, red cells, and platelets in midgestation
(E14.5) mouse embyros. We conducted our screens on fetal
tissues24,25 (liver and blood) rather than adult bone marrow to
(1) minimize loss of lethal recessive phenotypes before birth,
Genomic mapping and genotyping
DNA from (B6 ⫻ B10) F2 embryos was analyzed using a panel of
80 B6/B10 single nucleotide polymorphisms (SNPs) that on average span
the mouse genome at 20-Mb intervals. Full details of SNP marker design
and genotyping are listed in supplemental data.
Gene sequencing
Sequencing of candidate genes was to locate the causal ENU B6 nucleotide
substitution. DNA was prepared from an individual affected mouse and
primers designed for candidate genes to amplify all exons plus or minus
15 bp to cover splice junctions. Primers are available on request. Amplicons
were then sequenced by the Sanger method on a 3730xl capillary sequencer
(Applied Biosystems). This automated platform used Big Dye Terminator
chemistry Version 3.1 (Applied Biosystems). The raw trace files were
analyzed using Lasergene software (DNASTAR) against B6 reference
genome. Mutations were confirmed in a second affected person.
Biochemistry
The E308G mutation on c-Myb that included an N-terminal HA tag was
recreated using a polymerase chain reaction-based mutagenesis approach,
after which the mutant was cloned into BamHI and XhoI sites of pcDNA3.1
(Invitrogen). Reporter and glutathione S-transferase (GST) pull-down
assays to detect interaction between Myb and CBP KIX were performed as
described in supplemental data.
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BLOOD, 23 DECEMBER 2010 䡠 VOLUME 116, NUMBER 26
HSC GENE DISCOVERY BY ENU MUTAGENESIS
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Statistics
Data were analyzed for significance between groups using a 2-tailed
Student t test. Differences were considered significant at P ⬍ .05.
Results
A screen for hematopoietic stem/progenitor cells and blood
production
Our primary aim was to find recessive mutations causing abnormal
definitive HSC production or turnover. A secondary aim was
mutations that resulted in defective red cell and/or platelet production. Most of the knowledge about HSC frequencies and behavior
has come from various assays using adult bone marrow. However,
FL is a very rich source of HSCs that outcompete bone marrow
stem cells in most scenarios.26,27 To efficiently screen for long-term
HSC (LT-HSC) and progenitor cell phenotypes in FL, we first set
up a high-throughput FACS assay using wild-type C57BL/6J (B6)
mice (Figure 1B). We chose this strain for mutagenesis because we
have extensive experience in using it for ENU screens,11,14 the
mouse reference genome was built using B6 DNA making ENUgenerated SNP detection easier, and many monoclonal antibodies
have often been used specifically in B6. In particular, functional
HSC assays, such as competitive repopulation assays, are easier
using B6 congenic strains because of the availability of CD45
strain-specific antibodies.
Using an 8-color single-stain protocol, 6 functionally distinct
hematopoietic stem and progenitor cell subsets, LT-HSCs, multipotent progenitors (MPPs), common lymphoid progenitors (CLPs),
common myeloid progenitors (CMPs), granulocyte/macrophage
progenitors (GMPs), and megakaryocyte/erythrocyte progenitors
(MEPs), were quantified in parallel (Figure 1B). Self-renewing
LT-HSCs
were
gated
according
to
the
5-color
cKit⫹Lin⫺Sca1⫹CD150⫹flk2⫺ cell surface phenotype, whereas
MPPs were cKit⫹Lin⫺Sca1⫹CD150⫺flk2⫹.26,27 The 4 subsets of
lymphoid and myeloid progenitors were gated according to published protocols.28,29
By a separate 3-color FACS analysis and cell size measurement
on a log scale, we also established a companion high-throughput
screening assay for subsets of blood cells and platelets in fetal
blood (FB) based on recently published work (Figure 1C).3,5
YS-derived primitive red blood cells (RBCs) were identified based
on their large size, nucleus (ie, Hoechst 33342 positivity), and
expression of the cell surface marker TER119. FL-derived definitive RBCs were medium-sized and expressed TER119 but were
enucleated (Hoechst 33342-negative). Finally, platelets were very
small in size and stained for CD41 but not Hoechst 33342.
Together, these 9 subsets of cells assayed in parallel provided a
high-resolution snapshot of early hematopoiesis in both a primary
tissue and the periphery (Figure 1A). The use of 2 complementary
screening assays each with multiple phenotypic readouts also
provided internal controls to minimize the identification of false
positives.
Ontogeny of hematopoietic stem and progenitor cell
compartments
We analyzed FL and FB samples from B6 embryos aged E12.5,
E13.5, and E14.5 to examine the dynamics of stem and progenitor
cell proliferation. A 5-fold increase in nucleated cells in the FL was
observed with each developmental day (Figure 2A). Primitive
RBCs formed the majority (⬃ 80%) of the circulating FB at E12.5,
Figure 2. Ontogeny of hematopoietic compartments in FL and blood. (A) Total
numbers of viable nucleated blood cells in E12.5, E13.5, and E14.5 FLs. (B) Percentages of primitive RBCs, definitive RBCs, and platelets in E12.5, E13.5, and E14.5
fetal blood. (C) Percentages of LT-HSCs and 5 progenitor populations in E12.5,
E13.5, and E14.5 FLs. (D) Total numbers of HSCs and 5 progenitor populations in
E12.5, E13.5, and E14.5 FLs. E12.5 (n ⫽ 29), E13.5 (n ⫽ 31), and E14.5 (n ⫽ 38)
C57BL/6J embryos were analyzed in 3 separate experiments, with data from
1 representative experiment shown. Viable cells were determined by Trypan Blue
exclusion.
a frequency that dropped by approximately 20% with each
developmental day (Figures 1A, 2B). This is consistent with the
gradual switch from primitive to definitive erythroid cells in the
circulation.3,4 We found that primitive erythroid cells underwent
progressive enucleation in the circulation to greater levels than
realized until recently,3 such that by E14.5 approximately 20%
were enucleated (Figure 1C). The reduction in primitive RBCs was
mirrored by a relative increase in definitive RBCs from approximately 5% at E12.5 to approximately 25% at E13.5 and
approximately 50% at E14.5 (Figure 2B). By comparison, mean
platelets counts of approximately 0.5% to 1% relative to RBCs
were relatively constant across developmental days (Figure 2B),
consistent with substantial platelet production from the YS as
well as the FL.5
Frequencies (Figure 2C) and absolute numbers (Figure 2D) of
the 6 HSC/progenitor cell subsets in FL were quantified across
developmental days. MEP was the predominant progenitor
population, with the E14.5 frequency progressing
MEP ⬎ GMP ⬎ CMP ⬎ MPP ⬎ CLP ⬎ LT-HSC. LT-HSC, MPP,
and CLP were very low in number at E12.5, composing only a few
hundred cells but expanding to 5000 to 20 000 cells by E14.5.
Based on these data, the optimal developmental age to conduct
our screen was E14.5, when the primitive and definitive RBC
compartments in FB were at approximately 50% relative frequencies (Figures 1, 2), facilitating accurate quantification of both. In
addition, mice harboring targeted deletions of genes essential for
definitive hematopoiesis survive to approximately E14.5 because
the primitive wave can sustain embryos until then.8,30 Furthermore,
we reasoned that loss-of-function or hypomorphic alleles of genes
involved in both primitive and definitive hematopoieis, such as
Epo, EpoR, or SCL/tal-1, might be viable or recently dead at
E14.5.31 Lastly, E14.5 FL is large and easy to process, whereas
older FL is harder to dissociate into single cells.
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BLOOD, 23 DECEMBER 2010 䡠 VOLUME 116, NUMBER 26
Figure 3. Composition of hematopoietic compartments in FL and blood in inbred mouse strains. (A) Percentages of LT-HSC and 5 progenitor populations in FLs across
inbred mouse strains. (B) Percentages of primitive RBCs, definitive RBCs, and platelets across inbred mouse strains. E14.5 embryos from C57BL/6J (n ⫽ 24), C57BL/10SnJ
(n ⫽ 16), C57L/J (n ⫽ 14), BALB/b (n ⫽ 16), C3H/HeH (n ⫽ 24), CBA/CaJ (n ⫽ 17), FVB/NJ (n ⫽ 21), and 129/SvEv (n ⫽ 31) strains were analyzed in 3 separate
experiments, with data from 1 representative experiment shown.
HSC compartments in inbred mouse strains
Because an outcross to a non-B6 inbred strain is required to map
the causative ENU mutation underlying any phenotype of interest,
we needed to ensure that the FACS assays were robust and identical
in the mapping strain eventually chosen. Numerous phenotyping
surveys encompassing a broad range of biologic disciplines have
been conducted in inbred strains,32,33 but none has specifically
focused on the variability on HSC/progenitor cell surface profile34
and frequency35,36 across a comprehensive number of inbred mouse
strains, particularly with recent advances in identified HSC
markers.26,27
We analyzed 7 inbred strains (C57BL/10SnJ, C57L/J, BALB/b,
C3H/HeH, CBA/CaJ, FVB/NJ, and 129/SvEv) alongside our
ENU-mutagenized B6 stain for the baseline composition and
enumeration of HSC/progenitor cell subsets in FL, and erythroid
cells/platelets in FB. There were some major surprises. For
example, a clear lack of Sca1 cell surface expression in BALB/c,
C3H/HeH, and CBA/CaJ strains resulted in an inability to recognize and score LT-HSCs using Sca1 (Figure 3A; supplemental
Figure 1A). FVB/NJ and 129/SvEv strains also showed a reduction
in Sca1 expression relative to B6, C57BL/10SnJ, and C57L/J. One
possible explanation for this apparent absence of Sca1 surface
expression is that the epitope recognized by the Sca1 monoclonal
antibody may have amino acid changes because of functionally
conservative SNPs in non-B6 strains. Alternative antibodies raised
against different epitopes or polyclonal antisera could give differ-
ent results. Nevertheless, our choice of mapping strain to detect
HSC phenodeviants was limited to B6-related and FVB/N strains.
In addition, CLP frequency was similar to C57BL/6 levels in B6
variants and 129/SvEv mice, but all other strains had lower
frequencies (Figure 3A). In addition, a shift in CD34 and Fc␥R cell
surface staining resulted in ambiguous GMP frequencies for all but
C57BL/10SnJ, whereas the CMP/MEP subsets were less variable
across the strains tested (Figure 3A; supplemental Figure 1B).
FB was also tested (Figure 3B). Although precise developmental timing between mouse litters was one experimental variable that
sometimes caused skewed ratios between circulating primitive and
definitive RBCs (Figure 2), there was a distinct lack of TER119
expression on the surface of primitive RBCs of C3H/HeH and
FVB/NJ strains (supplemental Figure 2). TER119 expression on
definitive RBCs was more uniform across all mouse strains; a SNP
in the gene encoding the surface protein recognized by the TER119
monoclonal antibody is thus unlikely (supplemental Figure 2).
Based on these observations, we outcrossed our ENUmutagenized B6 pedigrees to the C57BL/10SnJ (B10) strain
because it showed the least variability in hematopoietic profile
relative to B6 across all parameters examined, an unsurprising
finding given their common ancestry37 and close position on the
mouse phylogenetic tree.38 Although this meant a reduction in
polymorphisms between these 2 strains and mapping power,39 we
thought this was superseded by the need to reliably identify
phenodeviants on outcrossing.
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BLOOD, 23 DECEMBER 2010 䡠 VOLUME 116, NUMBER 26
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Table 1. ENU mutant pedigrees
Name (English
translation)
Phenotype
Fetal liver
Blood
Map location
Mutation
c-Myb A923G
Booreana (white)
Increased HSC
Anemia and increased platelets
Chromosome 10: 33-57 Mb
Kandarra (blood)
Normal at E13.5
Anemia at E13.5
Chromosome 2: 67-93 Mb
Undetermined
Mulkirri (plenty)
Normal
Severe anemia, normal platelets
Chromosome 7: 81-115 Mb
Undetermined
Undetermined
Chokerre (red)
Increased HSC
Anemia and increased platelets
Backcrossing
Kamu (blood red)
Normal
Anemia
Backcrossing
Undetermined
Wonggan (liver)
Increased HSC
Normal
Backcrossing
Undetermined
Summary of ENU pedigrees screened
We chose a concentration of ENU based on many years of
experience in the Australian Phenomics Facility. Previous experiments have determined that we generate approximately 30 mutations in coding genes in each founder G1 male using a standard
3 ⫻ 90 mg/kg ENU.40 Using our FACS assays, we screened 1375
G3 embryos at E14.5 from 45 B6 ENU pedigrees and identified 6
strains with phenotypes in HSC/progenitors and/or early blood cell
production. Supplemental Table 1 documents the first 1107 embryos and 34 pedigrees screened. Average litter size was 8 and the
embryo resorption rate was approximately 15%. Some pedigrees
showed a consistently higher rate of embryo resorption possibly
indicative of either a dominant or recessive early embryonic lethal
phenotype. We found 6 ENU pedigrees with reproducible defects
defined as present in at least 3 independent litters. ENU11.21
(kandarra), ENU11.28 (booreana), and ENU11.211 (mulkirri)
have been outcrossed and heritability confirmed. A further 3
pedigrees, ENU11.208 (chokerre), ENU11.235 (wonggan), and
ENU11.312 (kamu), are in the process of outcrossing for mapping
(Table 1).
Identification of the Booreana mutant phenotype
The ENU strain booreana (from the Aboriginal word for white)
was so named because embryo 8 from the first litter of pedigree
Figure 4. Booreana ENU mutant identification and heritability testing. Example of a variant embryo (mouse G3.8 from pedigree ENU11.28; A) identified by the FL screen
as having atypical stem cell and progenitor FACS hematopoiesis (increased LT-HSC, MPP, CLP, and CMP; decreased GMP and MEP; B) at E14.5. The C57BL/6J G1 male
founder was outcrossed to a C57BL/10SnJ female and then established as a true-breeding strain by intercrossing F1(B6 ⫻ B10) siblings and testing with the same screening
protocol. Additional variant embryos (mutant F2.2, F2.3, F2.4, F2.12, and F2.19) showed an identical abnormal increase in KLS (cKit⫹Lin⫺Sca1⫹) and other stem cell subsets (C-D),
and a perturbed fetal blood FACS profile (increased primitive RBC and platelets; decreased definitive RBC; E) at E14.5. Red dots represent putative recessive mutants; and
black dots, phenotypic wild-type littermates.
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BLOOD, 23 DECEMBER 2010 䡠 VOLUME 116, NUMBER 26
Figure 5. The booreana ENU strain harbors a
point mutation in the transactivation domain of
c-Myb. (A) Homozygous booreana mutants (boo/
boo) appear normal at E14.5. (B) Resequencing of
c-Myb (ENSMUSG00000019982) from 2 phenotypepositive embryos identified an A-to-G transition in
both cases in exon 8 at base pair 923, changing
amino acid 308 from glutamic acid to glycine. (C) Domain structure of c-Myb showing the E308G point
mutation in the transactivation domain. DNB indicates DNA binding; TA, transactivation/acidic; and
NRD, negative regulatory. (D) Complete blood counts
of 8-week-old booreana homozygous and heterozygous mutants and wild-type littermate controls as
measured by ADVIA 2120 machine. *Statistically
significant differences (P ⬍ .05) between boo/boo
and ⫹/⫹ groups. **Significance between boo/boo
and both ⫹/⫹ and boo/⫹ groups. (E-F) Blood films of
12-week-old boo/boo mice (E) and boo/⫹ littermates (F).
The mutant animals have increased platelets, polychromasia (p), and Howell-Jolly bodies (arrowheads)
and basophilic stippling (s). For panels E and F,
images were generated using an Olympus BX51
microscope with a U PlansApo 60⫻ lens, 1.35 NA,
under Olympus imersion oil. Images were collected
using an Olympus DP70 camera and DP controller
software. Digital images were adjusted and labeled
using Photoshop CS4. (G) Reporter assays in
293T cells cotransfected with the myeloperoxidase
gene promoter linked to lacZ and various HA-tagged
c-Myb constructs, including wild-type (WT), L302A,
M303V, and E308G (Booreana) mutations within the
TA domain. Bars represent mean normalized relative
light units (RLU), and error bars represent SD of the
mean of triplicate biologic assays. (H) Western blot of
transfected 293T cells for the HA tag and for endogenous ␤-actin showing equivalent transfection efficiency. (I) GST pull-down assays showing interactions between GST-CBP-KIX (KIX) and in vitro
translated c-Myb mutants. Input (ipt) and bound
wild-type (WT), E308G, or L302A radiolabeled c-Myb
proteins eluted from the indicated GST fusions are
shown in top panel; the bottom panel shows the
presence of the relevant GST proteins, visualized by
Coomassie blue staining, in each binding reaction.
ENU11.28 (Figure 4A) showed an expansion of LT-HSCs (Figure
4B,D). Booreana homozygous embryos (boo/boo) are indistinguishable from littermates by inspection (Figure 5A). However, boo/boo
FL has an increased frequency of LT-HSC (4-fold), MPP (3-fold),
CLP (3-fold), variable CMP numbers, but decreased GMP (3-fold)
and MEP (2-fold; Figure 4B). That some cell subsets had expanded
and others reduced strongly suggested a bona fide phenodeviant.
The distinctive boo phenotype recurred in one embryo (no. 17) in
the third litter of pedigree ENU11.28 (supplemental Figure 3). In
this case, CMP numbers were normal, but the other subsets were
like those in the founder embryo.
We outcrossed the heterozygous boo/⫹ G1 founder male from
pedigree ENU11.28 to a wild-type B10 female and then established
booreana as a heritable breeding line by intercrossing F1(B6xB10)
siblings and testing with the same FL FACS screening protocol.
Fulfilling our expectation from baseline phenotyping of non-B6
strains (Figure 3), the introduction of B10 genome did not suppress
the booreana phenotype, which was observed at an expected
Mendelian ratio (5/20; Figure 4C). There was a consistent increase
in LT-HSC and CLP, with reduction in MEP and GMP. CMP were
variable because of difficulties in gating these cells accurately in
boo/boo embryos (supplemental Figure 5). FB from boo/boo
embryos showed an expansion in their primitive RBC subset and
dramatic reduction in their definitive RBCs (Figure 4E; supplemental Figure 4), consistent with the decreased MEP frequency.
However, boo/boo embryos showed a striking 5- to 10-fold
increase in circulating platelets (Figure 4E; supplemental Figure 4).
Booreana embryos harbor a mutation in the transactivation
domain of c-Myb
We generated a boo/boo DNA pool (n ⫽ 12) alongside an unaffected pool of ENU11.28 embryos (n ⫽ 20) and analyzed these
against a panel of 80 B6/B10 SNPs to map the underlying booreana
mutation to a 57-Mb region on chromosome 10 containing
approximately 300 genes. The proto-oncogene c-Myb resides in the
interval and was a strong candidate for mutation given previous
reports describing hematopoietic defects in mice harboring c-Myb
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BLOOD, 23 DECEMBER 2010 䡠 VOLUME 116, NUMBER 26
mutations.22 Resequencing of all c-Myb exons identified an A-to-G
transition at nucleotide 923 in exon 8 only in those embryos that
displayed the characteristic booreana phenotype (Figure 5B). The
DNA mutation results in a glutamic acid to glycine substitution at
amino acid 308 (E308G) in the transactivation (TA) domain of
c-Myb (Figure 5C). Interestingly, the c-MybE308G/E308G mutation is
only 5 amino acids from another ENU mutant, c-MybM303V/M303V
(also an A-to-G transition), which resulted multilineage hematopoietic abnormalities (see “c-Myb and hematopoiesis” below).23
To test the biochemical function of c-MybE308G/E308G, we cloned
the mutant cDNA into an expression vector and tested its ability to
transactivate the myeloperoxidase gene promoter in reporter assays. Compared with wild-type c-Myb, the E308G mutation is
completely inert (Figure 5G). There was slight residual function in
the M303V TA domain mutation, suggesting that the E308G
mutation is more severe. Because c-Myb has been reported to bind
p300 and CBP via its TA domain,23 we also asked whether the
mutation in the acidic residue disrupted interaction. We found
Figure 6. Reduced B lymphopoiesis and abnormal
myeloid differentiation and myelofibrosis in boo/
boo mice. (A-B) Hematoxylin and eosin stain of spleen
shows reduced white pulp and increased and disorganized red pulp with amorphous pink material in boo/boo
mice. (C-D) Perl stain showing increased iron accumulation in red pulp macrophages within boo/boo spleen.
(E-F) Increased reticulin (fibrosis) in boo/boo mice.
(G-H) Absent B cells and relatively reduced T cells in
boo/boo spleen. Numbers indicate percentage of cells
falling within the indicated FACS gates. (I-J) Increased
frequency of CD71⫹/TER119⫹ mature erythroid cells
and also CD71lo immature cells in boo/boo spleen.
(K-L) Increased Gr1hi neutrophils, Gr1int monocytes,
and TER119⫹ erythroid cells in boo/boo spleen.
(M-N) Pie graphs show percentage contribution of
B220⫹ B cells, CD3⫹ T cells, TER119⫹ erythroid cells,
Gr1 bright granulocytes, Gr1 intermediate macrophages, and “other” cells (mostly CD71 weak early
erythroid lineage cells) to the spleen. Numbers represent the mean of FACS analyses from 6 boo/boo mice
and 6 wild-type (⫹/⫹) littermate controls.
HSC GENE DISCOVERY BY ENU MUTAGENESIS
5855
complete absence of binding of CBP to E308G and L302A
mutations in GST pull-down assays in 293T cells (Figure 5I).
Adult c-MybE308G/E308G mice are a model for human essential
thrombocythemia with myelofibrosis
Like c-MybM303V/M303V mice, we found that 8-week old boo/boo
mice had markedly increased platelets (P ⬍ .001), anemia
(P ⫽ .003), and increased reticulocytes (P ⬍ .001; Figure 5D).
Boo/boo homozygotes also have neutropenia (P ⫽ .01), which was
not reported in c-MybM303V/M303V mice, suggesting some biochemical differences between these alleles. There were red cell abnormalities, including polychromasia and Howell-Jolly body inclusions
(Figure 5E-F). There was moderate splenomegaly in boo/boo
adults (254 ⫾ 46 mg, n ⫽ 6) compared with heterozygous littermates (115 ⫾ 15 mg, n ⫽ 5) because of expansion of the red pulp
(Figure 6A-B). On the other hand, the white pulp/lymphocyte
component of spleen was reduced in boo/boo mice. The overall
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5856
BLOOD, 23 DECEMBER 2010 䡠 VOLUME 116, NUMBER 26
PAPATHANASIOU et al
bone marrow
+/+
spleen
0.25
DRAQ5
DRAQ5
0.46
megs
Figure 7. Increased and aberrant megakaryopoiesis in boo/boo mice. FACS for CD41 and the DNA
intercalating dye DRAQ5 in bone marrow and spleen of
boo/boo and wild-type (⫹/⫹) littermates. There is a
marked increase in large CD41⫹ cells (megakarocytes)
in spleen (⬃ 20-fold) and bone marrow (⬃ 8-fold).
Ploidy analysis shows a “right shift” in boo/boo spleen
(ie, more megakaryocytes have ⬎ 8n ploidy and less
have 2n ploidy).
spleen
platelets
megs
Ploidy
2n
≥16n
8n
platelets
4n
2n
4n
8n
≥16n
0.46
0.88
CD41
DRAQ5
CD41
2n
4n
8n
≥16n
3.00
megs
platelets
4.62
CD41
7.47
DRAQ5
DRAQ5
boo/boo
56.32
26.89
9.17
6.35
megs
Ploidy
2n
≥16n
platelets
4n
8n
2n
4n
8n
≥16n
10.39
CD41
splenic architecture was disorganized in many cases with pink
amorphous material in the red pulp, increased iron staining, and
markedly increased reticulin staining (Figure 6C-F).
Megakaryocytes in boo/boo mutants were also increased approximately 8-fold in the bone marrow and approximately 20-fold in the
spleen as determined by CD41 staining (Figure 7). In addition to an
increase in number, there was an increase in nuclear ploidy of
boo/boo megakaryocytes as determined by DRAQ5 incorporation
in CD41⫹ cells (Figure 7). Together, these changes are very
reminiscent of human myeloproliferative neoplasms (MPNs) such
as essential thrombocythemia (ET) with myelofibrosis (MF; see
“Homozygous Booreana mice are a model for human essential
thrombocythemia and myelofibrosis” below).
Splenic FACS analysis of booreana adult mice revealed markedly reduced B-cell lymphopoiesis in homozygous mutants (Figure
6G-H), as well as an increased frequency of CD71⫹/TER119⫹ cells
(Figure 6I-J), Gr1hi neutrophils, Gr1int monocytes, and TER119⫹
erythroid cells (Figure 6K-L). Gr1int cells were confirmed to be
monocytes by coexpression of Ly6G (data not shown). The
percentage of splenic CD3⫹ T cells was reduced to approximately
50% of wild-type littermates (Figure 6M-N), but absolute splenic
T-cell numbers are probably close to equivalent given the 50%
increase in spleen weight.
Discussion
Forward genetics offers the potential to find novel genes involved
in any biologic process if a robust assay is established for reliable
detection of phenodeviants. Dominant and recessive screens using
high-throughput blood cell counting assays have successfully
identified mutations in many hematopoietic genes.11,14,22-25 Some
were well known from previous studies, whereas others were
DRAQ5
2n
4n
8n
≥16n
47.46
29.73
18.49
3.41
unexpected players. Embryonic recessive screens have simply
relied on identification of visible phenodeviants.24,25 To our knowledge, this is the first attempt to use quantitative FACS assays to
detect perturbations in all the major definitive HSC/progenitor cell
subsets. Such an undertaking would be difficult in adult mice, but
our father-daughter mating strategy and fetal screen of G3s have
enhanced the feasibility of such an undertaking; with a small team,
we have performed a pilot screen of 45 pedigrees and found
6 recessive mutations. The rapid cloning of a novel point mutation
in the c-Myb oncogene provides proof of principle that informative
mutations in key definitive stem cell genes can be identified by
using FACS and a recessive breeding strategy.
Strain differences in HSC and RBC antigen expression
In the process of searching for an appropriate mapping strain, we
uncovered differences in surface expression of stem cell markers.
For example, Sca1 is not expressed on the cell surface of HSCs in
several commonly used mouse strains, such as BALB/c, C3H, and
CBA, and is weak in 129/Sv. This has implications for using Sca1
for the study of HSC biology in mice generated from gene-targeted
ES cell lines from a 129/Sv genetic background. One possibly
trivial explanation is that the epitope recognized by the Sca1
monoclonal antibody (D7) is slightly divergent in the non-B6
strains because of an SNP that results in a conservative amino acid
change. Thus, Sca1 might be present in a variant form on HSCs in
these strains. Further studies with different hybridomas or polyclonal antisera to Sca1 would be necessary to resolve this.
The TER119 red blood cell epitope does not seem to be present
on embryonic red cells of some strains (C3H and FVB). TER119 is
associated with glycophorin A, but the precise epitope has not been
identified.41 Its absence in the primitive erythroid wave of certain
strains is intriguing; it is present in the definitive wave of these
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BLOOD, 23 DECEMBER 2010 䡠 VOLUME 116, NUMBER 26
strains so the genes coding for TER119 must be present in the
genomes, and the SNP argument is not likely to be valid in this
case. Our results suggest failure of TER119 detection in certain
strains is either the result of a lack of a key regulatory protein in the
primitive wave of these strains or of a partner protein or processing
pathway component, which normally leads to stable protein
accumulation at cell surface. These results suggest TER119 has
limited utility for the study of primitive erythroid cell differentiation in certain mouse strains.
HSC GENE DISCOVERY BY ENU MUTAGENESIS
5857
from receptors, such as EpoR and MPL.45 There are also reported
activating mutations in the cytoplasmic domain of the MPL
receptor (often at amino acid 515) in some cases of MF46 and other
described mutations, but in many cases of MPN the underlying
mutation is unknown. Our results suggest that it might be worth
resequencing c-Myb in cases of JAK2 V617F-negative and
MPL515-wild-type ET and MF. In addition, c-MybE308G/E308G mice
could be used to test new small molecules that inhibit JAKs or other
signaling pathways that are overly active in MPN, such as PDGF
receptor signaling in marrow fibroblasts.
c-Myb and hematopoiesis
A bottleneck on the pathway to gene identification
c-Myb is a well-known critical regulator of hematopoiesis and stem
cell biology in other organ systems.42 Gene knockout causes
embryonic lethality by E15.5 from anemia.8 Several ENU c-Myb
alleles have been discovered using screens to detect blood abnormalities. The M303V mutation impairs interaction with transcriptional coactivators, such as p300 and CBP.23 Our mutation in an
acidic residue of the TA domain leads to more severe transcriptional crippling and complete failure to interact with CBP. The
phenotype of adult c-MybE308G/E308G mice is probably slightly more
severe than the phenotype of c-MybM303V/M303V mice, consistent
with the in vitro gene reporter data. Interestingly, a mutation in the
KIX domain of p300, which disrupts interaction with c-Myb, leads
to a similar phenotype, suggesting that the c-Myb-p300/CBP
interaction is critical for aspects of hematopoiesis.43 Thus, because
the phenotype of the c-Myb knockout is much more severe than the
2 ENU strains, which disrupt p300/CBP cofactor binding, there
must be additional critical functions for c-Myb in vivo that do not
depend on traditional coactivator binding. For example, c-Myb
might have additional key roles as a transcriptional repressor or
work via additional protein interactions to direct gene expression or
chromatin architecture.
Carpinelli et al performed a dominant screen for perturbations
in platelet counts in adult mice.22 They found 2 alleles of c-Myb, which
they named plt3 and plt4 because they displayed mildly increased
platelet numbers in adult heterozygotes. c-Mybplt3/plt3 and c-Mybplt4/plt4
homozygote mice have very high platelet numbers (⬃ 4000 ⫻ 106/mL)
like c-MybE308G/E308G mice, and also mild anemia and reduced splenic
B cells. The plt3 mutation resides in the DNA-binding domain of
c-Myb, although it retains significant ability to transactivate a reporter
gene so is likely to retain some DNA binding. Only a fraction of
c-Mybplt3/plt3 mice survive the perinatal period so the phenotype is more
severe than the c-MybE308G/E308G or c-MybM303V/M303V phenotypes as one
might expect for a DNA-binding mutation. On the other hand,
c-Mybplt4/plt4 mice survive at Mendelian ratios from a c-Mybplt4/wt cross,
so is less severe than the DNA-binding mutation in plt3. The plt4
mutation results in mutation D384V within the negative regulatory
domain (NRD) domain of c-Myb. The in vivo interactions of this NRD
domain are still incompletely understood and probably shed important
knowledge about protein networks in which c-Myb operates.
We were able to identify the c-Myb mutation in booreana quickly
because the boo/boo phenotype was distinctive and previous
mutations and gene targeting of c-Myb had been reported.8,23
However, identification of the causative mutation in our other
pedigrees is taking longer for several reasons. First, there are
limited SNPs between B6 and B10, so there is limited power to find
meiotic recombinants in the intervals of interest. We have used
newly described SNPs derived from deep sequencing of the B10
genome (Bruce Beutler, The Scripps Research Institute, written
communication, September 2009) to limit the interval in mulkirri to
34 Mb on chromosome 7 and the interval in kandarra to 26 Mb on
chromosome 2 (Table 1). However, these large intervals include
558 and 641 coding genes in the intervals of mulkirri and kandarra,
respectively. Further meiotic recombinants will not help narrow the
interval because the bottleneck is in SNPs, not recombination
events. This is a consequence of using the B10 strain, but this is
unavoidable because of the strain specificity of our assays. We have
resequenced the coding exons of a few genes in these intervals,
which have a history in the hematopoietic field and are yet to find
the causative mutation in either strain. It is possible the causative
mutations reside outside coding regions (eg, promoters or enhancers), but this has been described rarely and is not the experience of
our colleagues.40 We think it is more likely the genes of interest are
novel with respect to hematopoiesis.
There has been a remarkably rapid advance in fast and cheap
deep sequencing technologies, which will aid mutation identification. These technologies are being used in large-scale cancer
resequencing projects but are also ideally suited to detecting ENU
mutations. One advantage of using B6 mice for mutagenesis is the
reference genome was generated using this strain, so unknown and
confounding mouse strain-specific SNPs are less likely to be
troublesome, and the ENU-induced mutation is more likely to be
obvious. There are different approaches in using these technologies, which are much cheaper than deep sequencing of the whole
mutant genome. We are currently using solution capture and
resequencing of coding exons within the mulkirri and kandarra
intervals as an efficient and relatively cost-effective way to find the
causative mutations.47
Homozygous Booreana mice are a model for human essential
thrombocythemia and myelofibrosis
Acknowledgments
c-MybE308G/E308G adult mice develop splenomegaly and a phenotype
reminiscent of the human MPN, ET, and MF. This is perhaps not
surprising because fibrosis in humans with these diseases is thought
to result from excess production of cytokines, such as PDGF via
increased megakaryocyte mass in bone marrow and spleen.44 In
approximately 50% of human cases, the underlying genetic defect
in ET and MF is a point mutation in the JAK2 kinase (V617F),
which leads to constitutive activity in the absence of stimulation
The authors thank Chris Goodnow and Ed Bertram for scientific
advice and financial support, Harpreet Vohra and Mick Devoy for
assistance with flow cytometry, and Shelley-Mae Bolton, Ryan
Dunstan, and Nadiah Roslan for animal husbandry.
This work was supported by the National Health and Medical
Research Council (C.J. Martin Fellowship; P.P.), the Leukemia
Foundation (scholarship; D.R.P.), and the University of Queensland (Postdoctoral Fellowship; P.Y.).
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5858
BLOOD, 23 DECEMBER 2010 䡠 VOLUME 116, NUMBER 26
PAPATHANASIOU et al
This article is dedicated to the memory of Jared Franklin Purton
(1976-2009).
Authorship
Contribution: P.P. conceived and designed the study, collected,
assembled, analyzed, and interpreted the data, composed figures,
wrote the manuscript, and gave final approval of manuscript; R.T.,
B.W., A.E.H., D.R.P., P.Y., S.O.C., and R.L. collected and assembled the data and gave final approval of the manuscript; T.J.G.
analyzed and interpreted data, edited the manuscript, and gave final
approval of the manuscript; and A.C.P. conceived and designed the
study, analyzed and interpreted the data, composed figures, wrote
the manuscript, and gave final approval of manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Peter Papathanasiou, Australian Phenomics
Facility, Australian National University, Acton ACT 0200, Australia; e-mail: [email protected]; or Andrew C. Perkins, Institute for Molecular Bioscience, University of Queensland,
St Lucia QLD 4067, Australia; e-mail: [email protected].
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2010 116: 5849-5858
doi:10.1182/blood-2010-04-269951 originally published
online July 7, 2010
A recessive screen for genes regulating hematopoietic stem cells
Peter Papathanasiou, Robert Tunningley, Diwakar R. Pattabiraman, Ping Ye, Thomas J. Gonda,
Belinda Whittle, Adam E. Hamilton, Simon O. Cridland, Rohan Lourie and Andrew C. Perkins
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