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Prepublished online as a Blood First Edition Paper on May 31, 2002; DOI 10.1182/blood-2002-04-1064.
HEMATOPOIESIS
From the Genetics and Molecular Biology Branch and
Genetic Disease Research Branch, National Human Genome Research
Institute, National Institutes of Health, Bethesda, MD, and Program in
Gene Function and Expression, University of Massachusetts Medical
School, Worcester.
Core-binding factor Core-binding factor Although CBF The crucial role of the CBF complex in hematopoiesis is underscored by
the observation that CBFB or CBFA2 are targeted
by chromosomal rearrangements in nearly 30% of individuals with acute myeloid leukemia (AML).16 The primary chromosomal
rearrangement involving CBFB is inv(16)(p13q22).
Inv(16) is associated with almost all cases of AML subtype
M4Eo and results in the fusion of CBFB with
MYH11, the gene for smooth muscle myosin heavy
chain.17 Previously, we used a knock-in strategy to
generate a mouse model in which Cbfb-MYH11 is
expressed under the control of the endogenous mouse
Cbfb gene.18 Chimeric mice derived from
embryonic stem (ES) cells targeted with the knock-in
Cbfb-MYH11 gene were used to assess the leukemogenic
potential of the fusion gene.19 Although the
Cbfb-MYH11 knock-in chimeras did not develop leukemia
naturally in the first year of life, most of the animals developed AML
within 3 to 5 months after treatment with the chemical mutagen,
N-ethyl-N-nitroso-urea (ENU). The dose of ENU
used was not sufficient to induce leukemia in wild-type chimeras. The
leukemia in the Cbfb-MYH11 chimeras was characterized
by the presence of myelomonocytic blasts and occasional eosinophils,
very similar to patients with AML M4Eo. These observations
suggested that although expression of Cbfb-MYH11 is
not sufficient for leukemogenesis, it is a necessary event in the
multistep process that gives rise to leukemias associated with inv(16).
Analysis of the contribution of ES cells with the
Cbfb-MYH11 knock-in gene in chimeric animals provided
evidence that Cbfb-MYH11 blocks differentiation of
the myeloid and lymphoid cells at the level of the c-kit+
progenitors, but does not affect erythroid maturation in
adults.19 Expression of the Cbfb-MYH11
knock-in gene in heterozygous embryos results in a severe defect in
definitive hematopoiesis, a phenotype similar to that observed in
embryos containing homozygous knock-out of either Cbfa2 or
Cbfb. In vitro, the CBFB-MYH11 gene
product, CBF Considering the critical role of Cbfb in normal
hematopoiesis and leukemogenesis it is important to further
characterize its expression in different hematopoietic cell
populations. Previous studies indicated that Cbfb is
expressed in the central nervous system, cranial nerve and dorsal root
ganglia, eyes, limb bud, somites, and ribs of mouse embryos, as
assessed by in situ hybridization.1,14 In adults,
Cbfb expression is considered to be ubiquitous
because it has been detected in most adult tissues and various cell
lines by Northern blot analysis.11,12 In this paper we
characterize the expression of Cbfb in embryonic and
adult hematopoietic tissues and dissect the specific hematopoietic
defects associated with CBFB-MYH11 expression, using a newly
created Cbfb-GFP knock-in mouse model.
Generation of Cbfb-GFP knock-in mice
The targeting construct was linearized at a unique NotI site
and transfected into ES cells by electroporation. Homologous recombinant clones were identified by Southern blot analysis of gDNA
isolated from individual G418/FIAU-resistant ES cell colonies. The DNA
was digested with either XbaI or NcoI, and the
blotted DNA was hybridized with probes, one internal to the targeting DNA vector (Hygro) and one external (probe 0.2C).18
NcoI digestion generates a 15.7-kb band from the wild-type
Cbfb allele that is detected with the 0.2C probe. The
correctly targeted Cbfb-GFP allele generates a 6.3-kb
band detected with the 0.2C probe. XbaI digestion generates
a 7.4-kb band from the targeted allele that is detected with the Hygro probe.
Genotype analysis
Western blot analysis Lysates from adult tissues or ES cells were prepared by resuspending 1 × 106 cells in NuPage lithium dodecyl sulfate (LDS) sample buffer with reducing agent (Invitrogen) and boiling the samples for 15 minutes. The proteins were separated by electrophoresis on NuPage 4% to 12% bis-tris gels in 2-N-morpholino ethane sulfonic acid (MES) running buffer and transferred onto nitrocellulose membranes using the semidry blotting system (Amersham, Piscataway, NJ). Membranes were probed with a 1:10 dilution from a monoclonal antibody specific for Cbf (amino acids
1-141),14 or a 1:5000 dilution from a polyclonal antibody
specific for multiple endocrine neoplasia 1 (MEN1; gift from S. C. Chandrasekharappa, National Institutes of Health, Bethesda, MD),
followed by a secondary antibody conjugated to horseradish peroxidase
(HRP). Enhanced chemiluminescence (ECL; Amersham) was used to detect
the antibody complexes.
Ter119+ and Ter119 Cell staining and flow cytometry Peripheral blood was obtained from anesthetized animals by cardiac puncture. Bone marrow was obtained by flushing femur and tibia with fluorescence-activated cell sorter (FACS) buffer (5% fetal calf serum [FCS] in phosphate-buffered saline [PBS]), followed by trituration through a 25-gauge needle. Bone marrow, spleen, and peripheral blood samples were incubated in ACK lysing buffer (Biowhittaker, Walkersville, MD) to lyse the erythrocytes prior to staining with antibodies. Bone marrow and peripheral blood were stained with phycoerythrin (PE)-conjugated antibodies to CD3 (17A2), B220 (RA3-6B2), Mac1 (M1/70), Gr-1 (RB6-8C5), Ter119 (Ly 76), and c-kit (2B8; BD Pharmingen, San Diego, CA). Additional B-cell staining was performed using the following antibodies purchased from BD Pharmingen as described previously23: PE-conjugated anti-human serum albumin (HSA; M1/69), anti-CD-43 (S7); biotinylated anti-HSA (M1/69), anti-BP-1 6C3 and anti-IgM; and allophycocyanin (APC)-conjugated B220 (RA3-6B2). For staining of megakaryocytes, unlysed bone marrow was resuspended in PBS containing 5% donkey serum. Two hundred nanograms sheep anti-human platelet glycoprotein (GP) IIb-IIIa antibody (Affinity Biologicals, Hamilton, ON, Canada) was used for staining 1 × 106 cells. The secondary antibody was PE-conjugated donkey anti-sheep immunoglobulin (1:200 dilution). Cells were isolated from lymph node, thymus, and spleen of 3- to 6-month-old mice by passage through a nylon mesh. Cells were stained with PE-conjugated antibodies to CD4 (RM4-5) and Cy-chrome-conjugated anti-CD8 53-6.7 (BD Pharmingen). Appropriate isotype controls were used in each experiment. Cells were
stained for flow cytometric analysis by incubating with 0.2 µg to 1 µg antibody per 1 million cells in ice-cold FACS buffer for 30 minutes. After washing, cells were resuspended in 200 µL FACS buffer.
The GFP signal was detected on FL-1 channel of FACScan (BD Biosciences,
San Diego, CA) or FACSCalibur (BD Biosciences). PE was detected
on FL-2, and Cy-chrome on FL-3 on the FACScan. For 4-color experiments,
APC was detected on FL-7 of FACSCalibur.
Lineage depletion and cell sorting of bone marrow was performed as described previously24 using purified antibodies to CD4, CD8, B220, Mac1, GR1, and Ter119 (Caltag Laboratories, Burlingame, CA). Biotinylated c-kit (ACK4-biotin) antibody and streptavidin-PE (BD Pharmingen) were used to stain bone marrow cells after lineage depletion. Fetal liver and AGM were dissected from 11.5- and 12.5-dpc embryos using standard techniques. The tissues were dissociated by trituration using a 25-gauge needle and passed through a nylon mesh. Methylcellulose colony-forming assays Adult bone marrow and 11.5-dpc fetal liver cells were washed and resuspended in Iscove modified Dulbecco medium (IMDM; Invitrogen) with 10% fetal bovine serum (FBS; Stem Cell Technologies, Vancouver, BC, Canada). Cells were incubated in 35-mm suspension dishes in IMDM containing 0.9% methylcellulose, 15% FBS, 1% bovine serum albumin, 10 µg/mL bovine pancreatic insulin, 200 µg/mL human transferring, 10 4 M 2-mercaptoethanol, 2 mM L-glutamine, 50 ng/mL recombinant murine stem cell factor (rmSCF), 10 ng/mL recombinant
murine interleukin 3 (rmIL-3), 10 ng/mL rmIL-6, and 3 U/mL recombinant
human erythropoietin (MethoCult GF M3434; Stem Cell
Technologies). Colonies were visualized and counted after 10 days in culture.
Generation of the Cbfb-GFP knock-in mouse model We previously demonstrated that Cbfb-MYH11 blocks differentiation of hematopoietic cells and promotes the development of AML in mice.18,19 To elucidate the normal role of Cbfb in hematopoiesis, and characterize the defect caused by Cbfb-MYH11, we generated mice expressing Cbf tagged with the GFP. The knock-in targeting construct
that contains Cbfb exon 5 (amino acids 1-151) fused in-frame to GFP cDNA is shown in Figure
1A. The fusion protein generated by this
construct maintained the ability to interact with Cbf2 in vitro and
exhibited a subcellular localization pattern that was identical to
wild-type Cbf in cultured cells (data not shown). We anticipated
that Cbf-GFP should function normally, at least with respect
to hematopoiesis, because a Cbfb (amino acids 1-141)
expression construct can rescue the hematopoietic defect in a
Cbfb null ES cell line.25 Southern blot
analysis demonstrated a 15% targeting efficiency and allowed
identification of several correctly targeted ES cell clones exhibiting
a 6.3-kb NcoI-digested band detected with the
external probe 0.2C (Figure 1B). To verify that the targeting vector
was integrated only once, we used a probe directed against the
hygromycin gene that is unique to the targeting vector to
demonstrate a single 7.4-kb band (Figure 1C). Western blot analysis
demonstrated expression of both the endogenous 25-kDa Cbf and the
47-kDa Cbf-GFP fusion protein in targeted ES cells (Figure 1D).
Three targeted ES cell clones (nos. 44, 52, and 74) heterozygous for
the knocked-in allele were injected into C57BL/6-derived host
blastocysts. Injection of ES cell clone 44 gave rise to low percentage
chimeras. Chimeric male mice from ES clones 52 and 74 were crossed with
129/Sv females and passed the targeted Cbfb-GFP
allele through the germline. All phenotypes were identical in adults
and embryos derived from either of the independently targeted clones.
Mice derived from both clones were used in these studies. There was no
significant difference in cell number or percentage of any
hematopoietic lineage in Cbfb+/GFP
compared with wild-type adults (data not shown). The studies in adult
mice were performed using heterozygous animals, whereas those in
embryos were done using both heterozygous and homozygous embryos.
Homozygous embryos died shortly after birth. The reason for the
neonatal lethality is unclear, but apparently unrelated to
hematopoiesis. The presence of functional stem/progenitor cells in
CbfbGFP/GFP embryos was confirmed by flow
cytometric analysis and methylcellulose colony assays of
stem/progenitor cells (Figure 4 and Table 2) and long-term repopulation
assays using 14.5-dpc fetal liver (data not shown). The presence and
normal distribution of all mature lineages was confirmed by flow
cytometric analysis of 16.5-dpc fetal liver and peripheral blood smear
of newborn CbfbGFP/GFP pups (data not
shown). These data suggest that hematopoiesis is relatively normal and
does not account for the lethality of the newborn pups.
Cbf in various hematopoietic cell
populations in adult mice, cells were harvested from several
hematopoietic tissues in Cbfb-GFP heterozygous
animals and analyzed for GFP expression by flow cytometry. FACS
analysis showed a single peak of GFP-expressing cells in the thymus,
lymph nodes, spleen, and peripheral blood, suggesting that most of the
cells in these tissues express Cbf (Figure
2A). By contrast, in the bone marrow
there were consistently 3 populations of nucleated cells that expressed
different levels of Cbf-GFP, ranging from no expression to high
levels of expression (Figure 2B, left panel). This was the first
indication that Cbf may not be expressed in all hematopoietic cell
populations.
Cbf -GFP expression in various lineages by flow cytometry. Analysis of GFP
expression in monocytes and granulocytes (Mac1+ or
GR1+ or both) in bone marrow (Figure 2B, middle panels) and
peripheral blood (data not shown) revealed single peaks of
GFP-expressing cells, indicating uniform expression of Cbf-GFP.
Megakaryocytes (GP IIb-IIIa+) also expressed a uniform
level of Cbf-GFP (Figure 2B, right panel). Nucleated
Ter119+ erythroblasts in the bone marrow did not express
Cbf-GFP. However, as shown in Figure 2C, as erythroid cells matured
from c-kit+ progenitors (R2) to Ter119hi
erythroblasts (R5), there was a progressive loss of Cbf-GFP expression. The majority of Ter119+ cells in the bone
marrow did not express Cbf-GFP. We confirmed that the Cbf-GFP
signal was representative of the normal distribution of Cbf by
examining endogenous Cbf expression in Ter119-enriched and
Ter119-depleted populations by Western blot analysis (Figure 2D, lanes
2 and 3). In a population that contained approximately 90%
Ter119+ cells, we were unable to detect endogenous Cbf
by Western blot analysis. By contrast, there was abundant Cbf
expression in the Ter119 population, as predicted by FACS
analysis. In addition, the levels of wild-type Cbf and Cbf-GFP
were comparable in the Ter119-depleted population from adult
Cbfb+/GFP bone marrow as assessed by Western
blot analysis (Figure 2D, lane 1).
The analysis of GFP expression in B220+ B lymphocytes in
the bone marrow revealed 2 populations (Figure 2E, left panel). The various stages of B-cell differentiation in the bone marrow and corresponding markers are reviewed by Hendriks et al.23
Figure 2E demonstrates that Cbf The number and percentage of CD4/8 T cells were normal in the thymus,
lymph nodes, and spleen of heterozygote Cbfb-GFP
animals, as was the percentage of CD3+ T lymphocytes in the
peripheral blood (data not shown). All of the populations expressed
uniform levels of GFP, suggesting that T lymphocytes express Cbf Cbf is expressed in hematopoietic stem
cells and progenitors. Previous studies have demonstrated that the
lineage-negative (Lin) c-kithi population of
cells in adult mice is significantly enriched for stem cells that can
support long-term repopulation of lethally irradiated animals, whereas
the Lin/c-kitlo population contains only
hematopoietic progenitors.24
Cbfb+/GFP mice had comparable numbers of
Lin cells as wild-type animals. Lineage depletion
enriched for GFP+ cells as evidenced by the increased ratio
of GFP+ to GFP cells in the Lin
population (3:1) compared to that in total bone marrow (2:1; Figure
3A). Closer examination revealed that the
entire population of Lin/c-kithi and
Lin/c-kitlo cells expressed Cbf-GFP
(Figure 3A, right panel). This suggests that a population enriched for
long-term repopulating hematopoietic stem cells and hematopoietic
progenitors expresses Cbf. A methylcellulose colony assay was used
as an additional method of examining the expression of Cbf-GFP in
progenitors. Bone marrow cells from heterozygous animals were sorted
into GFP+ and GFP populations (Figure 3B).
Equal numbers of cells (5 × 104) from each population
were plated in methylcellulose cultures containing SCF, IL-3, IL-6, and
erythropoietin. There was a more than 10-fold enrichment in erythroid
burst-forming units (BFU-Es), granulocyte-macrophage colony-forming
units (CFU-GMs) and granulocyte-erythrocyte-macrophage colony-forming
units (CFU-GEMs) in the GFP+ population compared with the
GFP population, suggesting that most, if not all, of the
hematopoietic progenitor cells express Cbf (Table
1). It is interesting to note that the
greatest enrichment was observed in the CFU-GEMs, which originate from
a more immature progenitor that gives rise to both erythroid and
myeloid cells.
Cbf in embryonic
hematopoietic cells, we dissected the major sites of hematopoiesis
including the AGM, fetal liver, and yolk sac from 11.5-dpc embryos. The GFP signal in wild-type yolk sac cells was indistinguishable from heterozygous and homozygous embryos (data not shown). In the AGM and
fetal liver, c-kit marks the hematopoietic stem-progenitor cells. The
c-kithi cells in the AGM at 11.5 dpc comprise 1% to 2% of
cells in the AGM, and all of them expressed Cbf-GFP (Figure
4A). In the fetal liver, the
c-kithi cells included 30% to 40% of the cells, and
again, all expressed Cbf-GFP, although in heterozygous animals, the
distinction between GFP+ and GFP was not as
clear as in the homozygous animals (Figure 4B). Nevertheless, sorting
the c-kithi cells from a heterozygous embryo into
GFP+ and GFP populations (Figure 4C) resulted
in a significant enrichment of erythroid (6- to 7-fold), myeloid (3- to
4-fold), and mixed (4- to 5-fold) CFUs, suggesting that myeloid and
erythroid progenitor cells express high amounts of Cbf (Table
2).
There was no significant difference in the percentage of c-kithi cells in the fetal liver and AGM of wild-type, heterozygous, and homozygous embryos (Figure 4A,B) nor was there any significant difference in the colony-forming potential of the fetal livers isolated from these animals (Table 2), suggesting that the hematopoietic stem cells and progenitors in homozygous embryos are intact. The c-kithi population of cells is absent from AGM and fetal liver of embryos expressing Cbfb-MYH11 Previous studies revealed that heterozygous (Cbfb+/MYH11) embryos expressing Cbfb-MYH11 exhibited a complete absence of definitive hematopoiesis in the fetal liver. To further characterize the defect in these embryos, we examined the expression of c-kit and Cbf-GFP in
the Cbfb+/MYH11 embryos. In the fetal
liver at 11.5 dpc, we found a complete absence of the
c-kithi (CD34+ and CD34)
population suggesting that expression of Cbfb-MYH11
prevented the formation or migration of the stem-progenitor cells
(Figure 5A,B). There were very few cells
expressing Cbf-GFP in the
CbfbGFP/MYH11 embryos, confirming the
absence of cells expressing Cbfb (and presumably
Cbfb-MYH11). In the AGM, the c-kithi
population represents the cells in the hematopoietic clusters that give
rise to the hematopoietic stem cells and progenitors.26 This population of cells was also absent from embryos expressing Cbfb-MYH11 (Figure 5C), suggesting that the defect
occurs very early in hematopoietic differentiation, prior to migration
of hematopoietic stem cells and progenitors from the AGM to the
fetal liver.
The importance of Cbfb in hematopoiesis and
leukemogenesis prompted us to investigate the expression pattern of
Cbf In this study, adult Cbfb-GFP heterozygotes were used
to analyze the expression of Cbf The importance of Cbf The only hematopoietic cells that do not express Cbf In heterozygous embryos expressing knocked-in
Cbfb-MYH11, histologic analysis of fetal liver prior
to death of the embryos by hemorrhaging revealed an absence of
definitive hematopoiesis. In vitro differentiation of fetal liver from
these animals resulted in a 30- to 100-fold reduction in the number of
myeloid and erythroid colonies.18 In this study, we
demonstrated that the entire population of c-kithi
hematopoietic stem cells and progenitors in the AGM and fetal liver
expresses Cbf This study provides a detailed analysis of Cbf
Two articles recently described mouse Runx 3 (Cbfa3) knock-out models.29,30 The data showed that Runx 3 may regulate proliferation and apoptosis of gastric epithelial cells, and may also act as a tumor suppressor in human gastric cancer. In addition, Runx 3 plays a critical role in the development of neurons in the cranial and dorsal root ganglia.
The authors would like to thank Darryl Leja for his help in formatting the figures.
Submitted April 8, 2002; accepted May 16, 2002.
Prepublished online as Blood First Edition Paper, May 31, 2002; DOI 10.1182/blood-2002-04-1064.
Supported by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation (M.K.) and The Leukemia and Lymphoma Society (L.H.C.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Pu Paul Liu, Room 3A18, Building 49, National Institutes of Health, 49 Convent Dr, Bethesda, MD 20892; e-mail: pliu{at}nhgri.nih.gov.
1. Adya N, Castilla LH, Liu PP. Function of CBFbeta/Bro proteins. Semin Cell Dev Biol. 2000;11:361-368[CrossRef][Medline] [Order article via Infotrieve]. 2. Komori T, Yagi H, Nomura S, et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. 1997;89:755-764[CrossRef][Medline] [Order article via Infotrieve]. 3. Otto F, Thornell AP, Crompton T, et al. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell. 1997;89:765-771[CrossRef][Medline] [Order article via Infotrieve]. 4. Mundlos S, Otto F, Mundlos C, et al. Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell. 1997;89:773-779[CrossRef][Medline] [Order article via Infotrieve]. 5. Lee B, Thirunavukkarasu K, Zhou L, et al. Missense mutations abolishing DNA binding of the osteoblast-specific transcription factor OSF2/CBFA1 in cleidocranial dysplasia. Nat Genet. 1997;16:307-310[CrossRef][Medline] [Order article via Infotrieve]. 6. Okuda T, van Deursen J, Hiebert SW, Grosveld G, Downing JR. AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell. 1996;84:321-330[CrossRef][Medline] [Order article via Infotrieve] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
