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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 108-117
Growth Disturbance in Fetal Liver Hematopoiesis of Mll-Mutant Mice
By
Hideshi Yagi,
Kenji Deguchi,
Atsufumi Aono,
Yoshihiko Tani,
Tadamitsu Kishimoto, and
Toshihisa Komori
From the Department of Medicine III, Osaka University Medical School,
Osaka, Japan.
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ABSTRACT |
The MLL (ALL-1, HRX) gene is frequently involved in chromosomal
translocations in acute leukemia and has homology with Drosophila trithorax, which controls homeobox gene expression and
embryogenesis. To elucidate the function of Mll, we generated mice with
a mutated Mll locus. Mice with a homozygous mutation were embryonic
lethal and died at embryonic day 11.5 to 14.5, showing edematous bodies and petechiae. Histological examination revealed that hematopoietic cells were decreased in the liver of homozygous embryos, although they
were composed of erythroid, myeloid, monocytic, and megakaryocytic cells with normal differentiation. Colony-forming assays using cells
from fetal livers and yolk sacs showed that the number of colonies was
markedly reduced and many of the colonies delayed to be recognized in
Mllmu/mu embryos, although some of the colonies from
Mllmu/mu embryos developed similarly with that from
Mll+/+ and Mll+/mu embryos, suggesting
the delayed onset of the proliferation of hematopoitic precursors.
These data show that the hematopoietic precursors were greatly reduced
in mutant mice, and suggest that Mll functions as a regulator of the
growth of hematopoietic precursors.
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INTRODUCTION |
MLL (ALL-1, HRX) is located on band q23
of chromosome 11 and involved in reciprocal translocations with at
least 25 chromosomal loci.1-4 These translocations are
frequently observed in acute leukemia, including acute lymphoblastic
leukemia, acute myeloblastic leukemia, acute myelomonocytic leukemia,
and acute monocytic leukemia, and the prognosis of these patients is
poor.1 The breakpoints of 11q23 are concentrated in a
restricted region of Mll, exon 8-14.5,6 Partial duplication
and interstitial deletion of Mll have also been reported in acute
leukemia.7,8
The Mll gene codes for a large protein, predicted to be 431 kD, that
shows homology with Drosophila trithorax4,6,9 and
is expressed in many organs and tissues including brain, cerebellum,
cerebral cortex, spinal cord, colon, liver, fetal liver, spleen,
thymus, tonsil, kidney, heart, thyroid gland, lung, testis, and
skeletal muscle in humans.6,10 Trithorax has a zinc finger
domain and is considered to have DNA-binding capacity.11,12 In Drosophila, trithorax controls the expression of homeobox
proteins and regulates embryogenesis.12,13 Mll also has an
AT hook, the domain that has DNA-binding capacity. In addition, Mll has been shown to be localized in nuclei, supporting the idea that Mll is a
DNA-binding protein.10,14 Further, CAT assay
has shown Mll to have a domain for transcriptional activation, which is located 3 of the translocation cluster region.15 The
Mll region encompassing amino acids 2829-2883 is a minimal
transactivation domain that showed 300- to 500-fold activation in
GAL4/CAT assay. It was suggested that the loss of this domain is
related to the leukemogenesis caused by chimeric proteins derived from
chromosomal translocations involving Mll.16
The function of Mll was previously analyzed in knockout mice that were
generated by disrupting exon 3B, which is located between the AT hook
and trithorax motifs.17 It was shown that heterozygously mutated mice showed homeotic transformations during embryogenesis and
hematological abnormalities including anemia and thrombocytopenia, and
that Mll controlled homeobox expression. Further hematological abnormalities were investigated by colony-forming assay of yolk sacs in
homozygously mutated embryos that died at embryonic day 10.5-11.5 (E10.5-11.5 ).18 These studies showed smaller numbers of
colonies from homozygously mutated yolk sacs than from wild-type and
heterozygous yolk sacs and demonstrated that colony-forming-unit granulocytes, erythroids, macrophages, megakaryocytes (CFU-GEMM), colony-forming unit-macrophages (CFU-M), and burst-forming
unit-erythroids (BFU-E) were markedly decreased in homozygously mutated
embryos.18 Another study using in vitro colony-forming
assay showed that double Mll knockout embryonic stem (ES) cells formed
a significantly greater number of hematopoietic colonies than normal ES
cells and that these colonies consisted of immature hematopoietic
cells.19 Further, chimeric mice carrying an Mll-AF9 fusion
gene found in t(9,11) developed tumors that were restricted to acute
myeloid leukemia.20 However, the function of Mll still
remains to be clarified.
We generated mutant mice of Mll locus by replacing the region including
exons 12-14 with PGK-neo to reveal the function of Mll. Because the
major breakpoint cluster region of Mll is restricted to the genomic
region of exon 8-14, 3 of exon 14 should code a major function
of Mll. Homozygously mutated mice died at E11.5-14.5. We discuss here
the hematological abnormalities of homozygously mutated mice.
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MATERIALS AND METHODS |
Construction of the targeting vector.
We screened the 129/Sv mouse genomic library in the Lambda Fix II phage
vector (Stratagene, La Jolla, CA) with an Mll cDNA fragment and obtained genomic fragments encompassing exons 11-16. After
making a restriction map and performing sequencing, we disrupted the
region including exons 12-14 by replacing it with a neomycin-resistant gene (neo) driven by a phosphoglycerol kinase (PGK)
promoter.21 We inserted a 1.3-kb
HindIII-HincII fragment 5 of exon 12 and a
1.6-kb EcoRI-BglII fragment of the PGK-neo cassette
into an Xba I site and an EcoRI-BamHI site of
the pBluescript SK (Stratagene), respectively, in which the Ssp
I fragment was replaced with the PGK-tk cassette as
described.21 To obtain the final targeting vector, we
inserted a 13.2-kb Kpn I fragment 3 of exon 14 into an
Sal I-Kpn I site of pBluescript SK containing 5
of exon 12, PGK-neo, and PGK-tk.
Generation of mutant mice.
The targeting vector was linealized by Not I digestion,
electroporated into E14-1 ES cells (1 × 107), and
selected with G418 (0.4 mg/mL) and gancyclovir (2 µmol/L). Resistant
colonies were selected, expanded, and screened for homologous recombination by Southern blot analysis. Screened ES cells heterozygous for the Mll mutation were injected into E3.5 blastocysts derived from
C57BL/6J, and the blastocysts were transferred into the uteri of
pseudo-pregnant ICR females. Chimeric mice were mated with C57BL/6J,
and germline transmission was confirmed by Southern blot analysis.
Brother-sister mating was then performed to generate homozygous
mutants.
cDNA synthesis and polymerase chain reaction (PCR).
Total RNA was prepared from whole embryos and livers at E12.5 using
LiCl/urea, and 1 µg of total RNA was reverse transcribed by Moloney
murine leukemia virus-reverse transcriptase (GIBCO-BRL, Rockville,
MD) in a 20-µL reaction volume, using random hexamers. PCR amplifications were performed with GeneAmp 2400 (Perkin-Elmer, Foster City, CA) and AmpliTaq DNA polymerase (Perkin-Elmer) in a
50-µL reaction volume, using 1 µL of cDNA solution and the
following primers: Mll-5, 5-AGTGGATGCCTTCCAAAGCC-3
and 5-GAGACCTGCTTGCTGGACTT-3; Mll-3,
5-CCAGCAGTGAGCATGTAGAG-3, and
5-TGAAGGCGGAAGCACTGCGT-3. Twenty cycles of amplification
were performed for the cDNA from livers (94°C for 30 seconds, 55 to
65°C for 30 seconds, 72°C for 30 seconds) using the following
primers at the indicated annealing temperatures: Hoxa10,
5-CTGGCCTCTGGCTCGACCGA-3 and
5-GTCCGTGAGGTGGACGCTAC-3 at 58°C; Hoxb3,
5-CCACCTACTACGACAACACC-3 and
5-TTGCCTCGACTCTTTCATCC-3 at 55°C; Hoxa7,
5-CAAAATGCCGAGCCGACTTC-3 and
5-CAGGGGTAGATGCGGAAACT-3 at 58°C; Hoxa9
5-GAGAATGAGAGCGGCGGAGA-3 and
5-AGACAGAAGGAGACGGACAG-3 at 55°C; Hoxc4,
5-CCACCACCACCCTGAGAAAT-3 and
5-TAACCTGGTGATGTCCTCTG-3 at 56°C; erythropoietin
receptor 5-TCATGTAGCCTGCACCAGGCTCCC-3 and
5-GTTGCTCAGAACACACTCAGTGCG-3 at 65°C; GATA1
5-CATTGGCCCCTTGTGAGGCCAGAGA-3 and
5-ACCTGATGGAGCTTGAAATAGAGGC-3 at 55°C. Amplified
products were electrophoresed and blotted to HybondN+ (Amersham,
Amersham Place, Bucks, UK). The membranes were hybridized with the
probes generated by PCR amplification.
Hematological analysis.
The total number of leukocytes and total red blood cells of wild-type
and heterozygous mice were determined at 4 weeks of age from blood
samples obtained from the eyes. Embryonic blood samples were collected
from the umbilical cord, cytocentrifuged on glass slides, and stained
with May-Grünwald/Giemsa.
Histological analysis.
For histological analysis, formalin-fixed paraffin-embedded sections of
embryos were stained with hematoxylin and eosin (HE) using standard
techniques.
Methylcellulose colony-forming assay.
Fetal liver cells were obtained from E12.5 embryos and dissected by
mechanical manipulation. Cells were suspended in  minimum essential
medium (MEM) (GIBCO-BRL), and the number of viable cells was
examined by tripan-blue staining. A total of 3 × 104
cells were cultured with 20% fetal calf serum (FCS), 1.2%
methylcellulose, 2-mercaptoethanol (100 µmol/L), and cytokines (human
erythropoietin [hEPO] at a final concentration of 2 U/mL, human
granulocyte colony-stimulating factor [hG-CSF] at 10 ng/mL, human
interleukin-6 [hIL-6] at 100 ng/mL, mouse granulocyte-macrophage CSF
[mGM-CSF] at 100 ng/mL, mouse stem cell factor [mSCF] at 100 ng/mL,
and mouse IL-3 [mIL-3] at 500 U/mL). After incubation in a fully
humidified atmosphere of 5% CO2 and 95% air at 37°C
for 7 days or 14 days, colonies were counted under a microscope. Yolk
sac cells were obtained from E10.5 embryos and dissected by mechanical
manipulation. Cells were suspended in MEM, and the number of viable
cells was examined by tripan-blue staining. The cells were cultured
under conditions described previously.22
Flow cytometric analysis.
Single-cell suspensions from fetal livers at E12.5 were prepared using
phosphate-buffered saline (PBS) and 4% FCS. These suspensions were
depleted of red blood cells using 0.14 mol/L NH4Cl in 17 mmol/L Tris (pH 7.3) before staining. Cells (5 × 105)
were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-Gr-1 and phycoerythrin (PE)-conjugated anti-Mac1 (PharMingen, San
Diego, CA) and analyzed using a FACScan (Becton
Dickinson). Dead cells were excluded on the basis of
propidium iodide uptake, and 10,000 events were acquired.
Northern blot analysis.
Total RNA was prepared from whole embryos and livers at E12.5. Ten
micrograms of RNA was electrophoresed, blotted to HybondN+ (Amersham),
and hybridized with a 32P-labeled 0.4-kb Xba
I-PvuII fragment of mouse retinoblastoma gene. Filters were
rehybridized with a 32P-labeled 0.85-kb fragment of mouse
GAPDH.
Western blot analysis.
Whole fetal body lysate from E12.5 embryos was electrophoresed in 12%
sodium dodecyl sulfate (SDS)-polyacrylamide gels, and the gels were
transferred to Immobilon-P (Millipore, Bedford, MA) with a semi-dry
blotting apparatus. After blocking the membrane with nonfat milk, the
membranes were incubated with anti-cdk2, anti-cdk4, anti-cyclinD1, and
anti-cyclinD3 antibodies (Transduction Laboratories Inc, Lexington,
KY). For detection of the first antibodies, we used ECL Western blot
analysis (Amersham).
In situ hybridization.
Riboprobes for in situ hybridization were prepared using a DIG RNA
labeling kit (Boehringer Mannheim GmbH Biochemica, Mannheim, Germany) according to the manufacturer's instructions.
For the Rb probe, a cDNA fragment was amplified by reverse
transcriptase (RT)-PCR using primers
5-ACTCTGGGGCATCTGCATCT-3 and
5-TCTTCTGGGTGTTCGAGGTG-3, and inserted in the
pBluescriptKS( ) (Stratagene). Hybridization was performed as
discribed.23
Whole-mount immunohistochemistry.
Embryos were fixed in 4% paraformaldehyde and stored in methanol at
20°C. For staining, embryos were incubated about 30 minutes with methanol containing 50% dimethyl sulfoxide (DMSO), and washed by
methanol containing 50% DMSO and 2% Triton X-100. The
embryos were blocked for 10 minutes by 1% periodate, and washed by
Tris buffer saline (pH 7.4). The embryos were blocked one overnight by
5% nonfat milk, and stained overnight by F4/80 rat anti-mouse macrophage antibody (ICN/Cappel, Costa Mesa, CA). The embryos were
washed for 6 hours and incubated in peroxidase-conjugated anti-rat IgG
second antibody (Cedarlane, Ontario, Canada). The embryos were washed
for 6 hours and detected using 0.1 mg/mL diaminobenzidine for 1 hour,
followed by 20 minutes' exposure to the same diaminobenzidine solution
containing 0.03% hydrogen peroxidase. The developed embryos were
stocked in 2:1 benzylbenzoate:benzylalcohol.
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RESULTS |
Generation of mutant mice of Mll locus.
The Mll gene, which spans 100 kb and contains at least 37 exons, has an
AT-hook, methyltransferase homology region, zinc finger domain, and two
trithorax homology regions. At the 11q23 translocation, Mll has a break
cluster region that is located at exons 8-14. Because we considered the
function of Mll to be dependent on the 3 of exon 14, we
constructed a targeting vector in which the region containing exons
12-14 was replaced with PGK-neo (Fig 1A). The targeting vector was electroporated into an E14 line of ES cells
and selected by G418 and gancyclovir. Targeted ES cells were injected
into blastocysts of C57BL/6J mice. The chimeras were mated with
C57BL/6J mice, and the Mll mutation was transmitted through the
germline.
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| Fig 1.
Targeted disruption of Mll. (A) Restriction map of
wild-type allele (a), targeting vector (b), and mutant allele (c).
Exons, PGK-neo, and PGK-tk are depicted as closed boxes. Exons are
numbered according to human MLL exons previously
reported.40 Arrows indicate the transcriptional direction
of PGK-neo. H, HindIII; Hc, HincII; K, Kpn I;
RV, EcoRV; X, Xho I. (B) Southern blot analysis of
fetal DNA. Genomic DNA isolated from embryos was digested with
EcoRV and hybridized with the probe shown in (A). Bands are
indicated corresponding to wild-type (12.5 kb) and mutant (3.5 kb)
genes. Wild-type (+/+), heterozygous (+/mu), and homozygous
(mu/mu) genotypes are shown. (C) RT-PCR analysis of Mll transcripts
from F2 fetus. The primers of Mll-5 and Mll-3 are
indicated by arrows in (D). The numbers of exons are shown in boxes.
Deleted exons are depicted as shaded boxes.
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After the intercrossing of heterozygous mice, the genotypes of their
litters were examined at 4 weeks of age by Southern blot analysis (Fig
1B), and no homozygous mice were found. Because we did not observe the
death of mice until 4 weeks of age, homozygous mice seemed to be lethal
during gestation. To determine when embryonic lethality occurred, we
analyzed embryos at various stages of gestation (Table 1). At E14.5, we observed no live
homozygous embryos. At E12.5, some of the homozygous embryos were dead,
as ascertained by the absence of heartbeat at the time of dissection.
Therefore, we concluded that homozygous embryos died at E11.5-14.5. The
appearance of homozygous embryos at E12.5-13.5 was edematous, with
bleeding apparent under the skin (Fig 2).
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| Fig 2.
Appearance of embryos at E13.5. (A) Wild-type littermate.
(B) Appearance of the homozygous Mll embryo. Subcutaneous edema and
hemorrhage are seen.
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We determined by RT-PCR whether the region of exons 12-14 was deleted
in homozygous (Mllmu/mu) embryos (Fig 1C and D). We
detected the transcript of 5 of exon 8, where methyltransferase
is located, in Mllmu/mu embryos as well as in wild-type
(Mll+/+) and heterozygous (Mll+/mu) embryos.
However, we did not detect the transcript of 3 of exon 11 in
Mllmu/mu embryos except for two aberrant transcripts, which
were found to be out of frame from our examination of the sequences
(data not shown).
Hematological abnormalities in heterozygous mice.
The numbers of red blood cells and white blood cells in the peripheral
blood of Mll+/+ and Mll+/mu littermates were
examined at 6 weeks of age (Table 2). The
Mll+/mu mice had mild anemia compared with
Mll+/+ littermates. However, the number of white blood
cells of Mll+/mu mice was not significantly different from
Mll+/+ littermates.
Histological examination of fetal livers.
Fetal livers from Mll+/+, Mll+/mu, and
Mllmu/mu littermates at E11.5 and E12.5 were examined
histologically. Fetal liver cells, particularly hematopoietic cells,
were slightly reduced in Mllmu/mu embryos compared with
those in Mll+/+ and Mll+/mu littermates at
E11.5 (Fig 3A and B). The decrease in
hematopoietic cells was prominent in the livers of Mllmu/mu
embryos compared with those of Mll+/+ and
Mll+/mu littermates at E12.5 (Fig 3C through F). In
addition, the total number of cells in Mllmu/mu fetal
livers was decreased at E12.5 in comparison with those of
Mll+/+ and Mll+/mu littermates
(Table 3). However, the appearance of fetal
liver cells was similar among Mll+/+, Mll+/mu,
and Mllmu/mu littermates in the liver stamp specimens
stained with May-Grünwald/Giemsa. Erythroid, myeloid, and
monocytic cells were present at various stages of differentiation in
Mll+/+, Mll+/mu, and Mllmu/mu fetal
livers (Fig 4A through D,
Table 4). Megakaryocytes were also present
in Mllmu/mu fetal livers (Fig 4E and F, Table 4). The
presence of granulocytes and monocytes in Mllmu/mu fetal
livers was also confirmed by fluorescence-activated cell sorter (FACS)
analysis using anti-Mac1 and anti-Gr1 antibodies (Fig 5). We also confirmed the presence of
macrophages in tissues of Mllmu/mu embryos by
immunohistochemistry using the antimacrophage antibody F4/80
(Fig 6, see page 110). Further, we analyzed
the components of peripheral blood at E12.5 and found mostly nucleated
red blood cells, with few leukocytes and a few anucleated red blood
cells derived from definitive erythropoiesis in fetal liver. These
findings were similar among Mll+/+, Mll+/mu,
and Mllmu/mu embryos (Fig 7,
see page 115).
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| Fig 3.
Histological examination of fetal livers. (A and B)
Sections of fetal liver at E11.5 stained with HE (original
magnification [OM] × 200). Hematopoietic cells in
Mllmu/mu fetal liver were slightly decreased. (C through F)
Sections of fetal liver at E12.5 stained with HE (OM × 100). The
decrease of hematopoietic cells in Mllmu/mu was prominent.
(A, C, and E) Mll+/+; (B, D, and F)
Mllmu/mu.
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| Fig 4.
Stamp specimens of fetal livers at E12.5 stained
with May-Grünwald/Giemsa. (A and B) Arrows indicate
granulocytes. (C and D) Arrows indicate monocytic cells. (E and F)
Arrows indicate megakaryocytes. OM × 200. (A and E)
Mll+/mu; (C) Mll+/+; (B, D, and F)
Mllmu/mu.
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| Fig 5.
FACS analysis of fetal liver cells at E12.5. Fetal liver
cells are stained by anti-Gr-1 and anti-Mac1 antibodies. Numbers in
rectangles indicate percentages of Mac1+Gr-1 cells and
Mac1+Gr-1+ cells.
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| Fig 6.
Detection of macrophages in Mllmu/mu embryos
by whole-mount immunohistochemistry using antimacrophage antibody
F4/80. The heads of embryos at E12.5 are magnified (OM × 80). There
are many F4/80+ cells (arrows). (A)
Mll+/mu; (B) Mllmu/mu.
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| Fig 7.
Peripheral blood smears at E12.5 stained with
May-Grünwald/Giemsa. Arrows indicate anucleated red blood cells
derived from definitive hematopoiesis. (A) Mll+/mu; (B)
Mllmu/mu.
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Colony-forming assays of fetal livers and yolk sacs.
To investigate the reason for the decrease in hematopoietic cells in
the livers of Mllmu/mu embryos, we performed a
colony-forming assay using E12.5 livers. Colonies were counted under a
microscope after 7 and 14 days of culture. After 7 days of culture, the
average number of hematopoietic colonies per 3 × 104
liver cells of Mllmu/mu embryos was about one fifth that of
Mll+/+ and Mll+/mu embryos
(Fig 8A), although the number of colonies
from Mllmu/mu liver cells was increased after 14 days of
culture (Fig 8B). As the number of liver cells in Mllmu/mu
embryos was about half of that in Mll+/+ and
Mll+/mu embryos, the number of colonies per liver from
Mllmu/mu embryos was much less than that from
Mll+/+ and Mll+/mu embryos even after 14 days
of culture. The colonies were smaller and fewer than those of
Mll+/+ and Mll+/mu embryos at 7th day of
culture, and many small colonies had appeared at 14th day of culture in
Mllmu/mu embryos. It was contrary to the observation in
Mll+/+ and Mll+/mu embryos at 14th day of
culture, in which most colonies were large (Fig 9, see page 115). Further, we
performed the colony-forming assay using cells from fetal liver at
E13.0 and assessed colonies morphologically
(Table 5). The number of colonies in
Mllmu/mu livers was less than one tenth of that in
Mll+/+ and Mll+/mu livers even after 14 days of
culture. Therefore, the colonies of every lineage were decreased,
although the decrease of GM colonies was most prominent. In addition,
we performed a colony-forming assay using cells from yolk sacs at
E10.5. The number of colonies per yolk sac in Mllmu/mu
embryos was less than one fifth that of Mll+/+ and
Mll+/mu embryos after 7 days of culture (Fig 8C). Prolonged
culture for an additional 7 days also increased the number of colonies
from Mllmu/mu embryos, but the number was still less than
that from Mll+/+ embryos (Fig 8D).
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| Fig 8.
Colony-forming assay of fetal livers and yolk sacs. (A)
Colony-forming assay of 3 × 104 fetal liver cells of
Mll+/+, Mll+/mu, and Mllmu/mu
embryos at E12.5. The number of colonies of Mll+/+ (n
= 13), Mll+/mu (n = 26), and Mllmu/mu (n
= 7) after 7 days of culture is shown. (B) Colony-forming assay of 3 × 104 fetal liver cells of Mll+/+,
Mll+/mu, and Mllmu/mu embryos at E12.5. The
number of colonies of Mll+/+ (n = 8),
Mll+/mu (n = 22), and Mllmu/mu (n = 5)
after 14 days of culture is shown. (C) Colony-forming assay of yolk sac
of Mll+/+, Mll+/mu, and
Mllmu/mu embryos at E 10.5. The number of colonies of
Mll+/+ (n = 8), Mll+/mu (n = 8), and
Mllmu/mu (n = 5) after 7 days of culture is shown. (D)
Colony-forming assay of yolk sac of Mll+/+,
Mll+/mu, and Mllmu/mu embryos at E 10.5. The
number of colonies of Mll+/+ (n = 8),
Mll+/mu (n = 8), and Mllmu/mu (n = 5)
after 14 days of culture is shown. n, number of embryos analyzed. Bars
indicate standard deviations, and ( ) indicates the average of the
number of colonies. Statistically significant difference at #$<.001,
  +<.01, *<.05. Statistical analysis was performed by
Student's t-test and Welch's t-test.
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| Fig 9.
Examination of colonies derived from the colony-forming
assays using fetal liver cells at E12.5. (A, C, E, G, and I) The
appearance of the colonies at the 7th and (B, D, F, H, and J) at 14th
days of culture. Arrows indicate small colonies that had appeared at 14th day of culture in Mllmu/mu. (E through J) Magnified
views of single colony. (A, B, E, and F) Mll+/+; (C, D,
G, H, I, andJ) Mllmu/mu.
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Analysis of Hox gene family expression and erythroid hematopoietic
markers.
We investigated the expression of the Hox gene family including Hoxa9,
Hoxa10, Hoxa7, Hoxb3, Hoxb4, Hoxb6, Hoxb8, Hoxc4, Hoxc9, and Hoxb5.
RT-PCR using RNA from whole embryos at E12.5 showed similar Hox gene
expression among Mll+/+, Mll+/mu, and
Mllmu/mu embryos except for Hoxa7 and Hoxc9 (data not
shown). Hoxa7 and Hoxc9 expression was lower in Mllmu/mu
embryos than in Mll+/mu and Mll+/+ embryos. We
also examined Hox gene expression using RNA from fetal livers at E12.5
(Fig 10). The expression of Hoxa9,
Hoxa10, and Hoxa7 was decreased in Mllmu/mu fetal livers as
compared with Mll+/+ and Mll+/mu fetal livers.
We detected similar expression of Hoxb3, very low expression of Hoxb5,
Hoxb8, and Hoxb6, and no expression of Hoxc9 and Hoxb4 in all
Mll+/+, Mll+/mu, and Mllmu/mu fetal
livers. Hoxc4 expression varied among Mllmu/mu fetal
livers. We also examined the expression of Epo receptor (Epo-R) and
GATA1. The expression of Epo-R and GATA1 was lower in
Mllmu/mu fetal livers than in Mll+/+ and
Mll+/mu fetal livers (Fig 10). This finding is consistent
with the observation that hematopoietic cells were greatly reduced in
Mllmu/mu embryos compared with those in Mll+/+
and Mll+/mu littermates. However, the reduction of Hoxa9,
Hoxa10, and Hoxa7 expression in Mllmu/mu fetal livers was
more significant than that of Epo-R and GATA1.
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| Fig 10.
RT-PCR analysis of Hox genes and hematopoietic markers.
RNA was extracted from fetal livers at E12.5. Results from two
independent experiments are shown. +/+,Mll+/+;
+/mu, Mll+/mu; mu/mu, Mllmu/mu.
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Analysis of expression of cell-cycle-related genes.
We examined Rb expression by Northern blot analysis using whole-body
samples from E12.5 (Fig 11A). Rb
expression seemed to be lower in Mllmu/mu embryos than in
Mll+/+ embryos. The reduced expression of Rb in
Mllmu/mu embryos was more evident when it was examined
using RNA from fetal livers at E12.5 (Fig 11B). Further, the expression
of cdk2, cdk4, cyclinD1, and cyclinD3 was examined by Western blot
analysis using lysate from whole bodies at E12.5 (Fig 11C through F).
Their expression was similar among Mll+/+,
Mll+/mu, and Mllmu/mu embryos. Further, we
examined Rb expression in fetal liver by in situ hybridization. The Rb
expression was detected in the hematopoietic cells of
Mllmu/mu fetal livers as well as Mll+/+ fetal
livers (Fig 12).
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| Fig 11.
Cell-cycle-related gene expression. (A) Northern blot
analysis of Rb using RNA from whole embryos at E12.5. (B) Northern blot analysis of Rb using RNA from fetal livers at E12.5. (C) Western blot
analysis of cdk2 using whole-body lysate at E12.5. (D) Western blot
analysis of cdk4 using whole-body lysate at E12.5. (E) Western blot
analysis of cyclinD1 using whole-body lysate at E12.5. (F) Western blot
analysis of cyclinD3 using whole-body lysate at E12.5. +/+,
Mll+/+; +/mu, Mll+/mu; mu/mu,
Mllmu/mu.
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View larger version (154K):
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| Fig 12.
Examination of Rb expression in fetal liver at E12.5 by
in situ hybridization. Rb expression was detected in hematopoietic cells of Mllmu/mu livers as well as Mll+/+
livers. (A and B) OM × 100. (C and D) OM × 200. (A and C)
Mll+/+; (B and D) Mllmu/mu.
|
|
| |
DISCUSSION |
We generated mutant mice of Mll locus and studied their hematopoiesis.
The Mllmu/mu mice were embryonic lethal at E11.5-14.5,
although previous studies of Mll knockout mice found embryonic
lethality at E10.5.17 It is likely that the difference in
time of lethality was caused by a difference in the construction of the
targeting vectors, because exons 12-14 were deleted in our targeting
vector and exon 3b was deleted in the targeting vector previously
reported.17 It was recently reported that the N-terminus of
Mll protein is suspected to function in directing the Mll protein to
its target genes.24 Therefore, it may be that our
Mllmu/mu mice produced a truncated Mll protein coded by the
5 of exon 12 and still retained some functions of Mll, although
our repeated experiments to detect a truncated Mll protein have been
unsuccessful.
At E12.5-13.5, Mllmu/mu embryos appeared edematous and had
subcutaneous petechiae, although histological study did not show any abnormalities in the heart or blood vessels of Mllmu/mu
embryos. Further, the petechiae would not have been caused by thrombocytopenia because NF-E2 knockout mice that lacked platelets were
not embryonic lethal and the embryos did not show
hemorrhage.25 Details of the edema and homeotic
transformations of Mllmu/mu mice will be reported in a
separate paper (H.Y. and T.K., manuscript in
preparation).
Histological examination of the livers showed that hematopoietic cells
in Mllmu/mu embryos were markedly reduced but were composed
of erythroid, myeloid, and monocytic cells with normal differentiation.
There were also a few megakaryocytes in Mllmu/mu fetal
livers. The presence of monocytes and granulocytes in
Mllmu/mu fetal livers was also confirmed by FACS analysis.
In addition, macrophages in the tissues of Mllmu/mu embryos
were detected by immunohistochemistry using anti-mouse macrophge
antibody F4/80. Therefore, all lineages in hematopoietic cells without
a skewing to primitive hematopoietic cells were observed in
Mllmu/mu embryos.
By colony-forming assays using fetal livers as well as yolk sacs, we
observed a marked reduction of hematopoietic precursors and a delayed
colony formation in Mllmu/mu embryos. The difference of the
number of hematopoietic precursors between Mllmu/mu embryos
and their counterparts seemed to be increasing during development,
because the difference of colony formation was most prominent when we
used embryos at E13 although these embryos were not deteriorated. Some
colonies from Mllmu/mu embryos were similar in size with
those from Mll+/+ and Mll+/mu embryos at 7th
and 14th day of culture. However, many colonies that were not visible
at 7th day of culture were recognized at 14th day of culture in the
assay using Mllmu/mu embryos. Therefore, the delayed colony
formation observed in Mllmu/mu embryos is likely to be
caused by a delay in the onset of the proliferation of hematopoietic
precursors. We did not observe evident abnormalities in the
distribution of the lineages of hematopoietic cells such as colonies
with primitive hematopoietic cells. However, the disturbance of
differentiation into specific lineages has not been excluded despite
the nearly normal differentiation of hematopoietic cells observed in
Mllmu/mu livers, because GM colony formation seemed to be
severely reduced (Table 5). This observation is consistent with the
previous study which showed a lineage-specific decrease in CFU-GEMM and
CFU-M.18 They also found that
Mll / colonies were fewer in number, took
longer to develop, and contained fewer cells than their wild-type and
heterozygous counterparts.18 These findings are also
similar to our results of colony assay using fetal livers and yolk
sacs. Thus, the data from histological analysis of fetal livers and
colony formation assays showed that hematopoietic precursors were
greatly decreased and the growth of hematopoietic cells, especially
hematopoietic precursors, was disturbed in Mllmu/mu
embryos, implicating the role of Mll in the growth regulation of
hematopoietic precursors. However, RT-PCR analysis did not show much
difference in the expression of receptors for hematopoietic growth
factors including Epo-R, gp-130, common  receptor, and c-fms
(M-CSF receptor) among Mll+/+, Mll+/mu, and
Mllmu/mu embryos, indicating that these hematopoietic
growth factors are not responsible for the hematological abnormalities.
We investigated the expression of the Hox gene family including Hoxa9,
Hoxa10, Hoxa7, Hoxb3, Hoxb4, Hoxb6, Hoxb8, Hoxc4, Hoxc9, and Hoxb5,
because Mll has homology with the Drosophila trithorax, which
has a zinc finger domain and controls the expression of homeobox genes
during the embryogenesis of Drosophila.13 The expression of some of these genes has been reported in normal hematopoietic cells as well as malignant hematopoietic
cells.26-28 Hoxa9 is concerned with leukemogenesis in
humans,29 and Hoxa9-deficient mice showed hematological
abnormalities similar to those of Mllmu/mu
mice.30 Antisense oligomers of Hoxb3, Hoxb4, Hoxb5, and
Hoxb6 reduced colony formation of the hematopoietic
cells.31 The overexpression of Hoxb4 especially influenced
hematopoiesis, and Hoxb4 was suggested to regulate early hematopoietic
proliferation.32,33 The induction by
retrovirus of Hoxb6 expression in mouse bone marrow cells resulted in
immortality of the infected cells.34 Further, the
overexpression of Hoxb3 in hematopoietic cells blocked differentiation
to B-lymphoid cells, influenced T-cell development, and induced
granulopoiesis.35 We also examined Hoxc9 and Hoxa7
expression because it has been reported that these genes are not
expressed in Mll-deficient embryos.17 Apart from Hoxc9 and
Hoxa7, we detected similar Hox gene expression among
Mll+/+, Mll+/mu, and Mllmu/mu
embryos when we used RNA from total embryos. The expression of Hoxc9
and Hoxa7 was detectable not only by RT-PCR analysis but also by whole
mount in-situ hybridization in E12.5 Mllmu/mu embryos (data
not shown), although their expression levels were decreased in
Mllmu/mu embryos. When we used RNA from livers at E12.5, we
detected reduced Hoxa9, Hoxa10, and Hoxa7 expression by RT-PCR. These
Hox genes may be the candidates responsible for the growth retardation
of hematopoietic cells in Mllmu/mu embryos, because our
RT-PCR study detected more significant reductions in the expression of
Hoxa9, Hoxa10, and Hoxa7 than in the expression of Epo-R and GATA1 in
Mllmu/mu fetal livers. However, the possibility still
exists that other cell types than hematopoietic cells caused the
reduction of these Hox genes.
We also analyzed the expression of genes that control the cell cycle,
because the phenotype of Rb-deficient mice was similar to that of
Mllmu/mu mice and both showed retarded growth of
hematopoietic cells.36-38 In vitro examination using the
antisense oligomer of Rb showed that Rb is concerned with erythroid
differentiation.39 Rb expression was lower in
Mllmu/mu embryos than in Mll+/+ embryos,
especially when we used samples from fetal liver. Therefore, we
investigated Rb expression in the fetal liver by in situ hybridization. We detected Rb expression in hematopoietic cells of
Mllmu/mu fetal livers as well as Mll+/+ fetal
livers. Thus, we consider that the reduction of Rb expression in
Mllmu/mu fetal livers observed by Northern blot analysis is
caused by the decrease of hematopoietic cells in Mllmu/mu
fetal livers. We also examined other cell-cycle-related proteins, including cyclinD1, cyclinD3, cdk2, and cdk4 that regulate cell cycle
at the point G1 to S state, but they were expressed similarly among
Mll+/+, Mll+/mu, and Mllmu/mu
embryos.
We analyzed the hematological abnormalities of the mutant mice of Mll
locus. Our data suggested that Mll functions in the control of the
growth of hematopoietic progenitors. Although we observed the decreased
expression of HoxA cluster genes in Mllmu/mu livers, the
mechanism which causes the growth disturbance of hematopoietic cells
remains to be clarified. Indeed, further investigation will be needed
to elucidate the mechanism of leukemogenesis by the chromosomal
translocation involving Mll, because the different mutations of both
functional copies of Mll generated by us and another group did not lead
to the complete blockage of differentiation or the acceleration of the
growth of hematopoietic cells.
| |
FOOTNOTES |
Submitted October 27, 1997;
accepted March 4, 1998.
Address reprint requests to Toshihisa Komori, MD,
Department of Medicine III, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka, 565, Japan.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank M. Sato for advice on histological examination; Y. Higashi, T. Ogura, and N. Takahashi for the probes of Hox genes; M. Seto for
anti-Mll antibody; and T. Matsunashi for technical assistance. We thank
R. Hiraiwa and Y. Ishinishi for maintaining mouse colonies and M. Ooi
for secretarial assistance.
| |
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Abstract
Introduction
Abstract