|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 87-95
Definitive But Not Primitive Hematopoiesis Is Impaired in
jumonji Mutant Mice
By
Kenji Kitajima,
Mizuyo Kojima,
Kuniko Nakajima,
Shunzo Kondo,
Takahiko Hara,
Atsushi Miyajima, and
Takashi Takeuchi
From the Mitsubishi Kasei Institute of Life Sciences, Institute of
Molecular and Cellular Biosciences, The University of Tokyo, Tokyo,
Japan.
 |
ABSTRACT |
A novel gene, jumonji was identified by a mouse gene trap
strategy. The jumonji gene encodes a protein containing a
putative DNA binding domain. The mice homozygous for jumonji
gene with a BALB/cA genetic background show hypoplasia of the fetal
liver and embryonic lethality, suggesting impaired hematopoiesis. In the peripheral blood of jumonji mutant embryos, the number of fetal liver-derived definitive erythrocytes, but not yolk sac-derived primitive erythrocytes, showed a marked reduction, suggesting that
jumonji mutants die of anemia. The defects of definitive erythrocytes in jumonji mutants seemed to be caused by a
decrease in the numbers of multiple hematopoietic progenitors including colony-forming unit-spleen (CFU-S) in the fetal liver. However, hematopoietic stem cells (HSCs) in the fetal liver of jumonji mutants could reconstitute the hematopoietic system of lethally irradiated recipients. In the fetal liver, the jumonji gene is expressed in fibroblastic cells and endothelial cells, but not in
Lin /c-Kit+/Sca-1+ cells
known to include HSCs. These results suggest that an environmental defect induce the impaired hematopoiesis in the fetal liver of jumonji mutant embryos.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THE JUMONJI (jmj) gene
and jmj mutant mice were obtained by a mouse gene trap
strategy.1 The mouse jmj gene maps at chromosome
13.2 The jmj gene encodes a protein that is
partially homologous to the human retinoblastoma-binding protein RBP-2, and to a putative protein encoded by human gene XE169, which escapes X-chromosome inactivation.1 In addition, the Jmj protein
contains nuclear localization signals, and a region homologous to the
AT-rich interaction domain (ARID) identified in the DNA binding protein Dead Ringer in Drosophila, in transcription factor Bright in
mouse, and in SWI1 in yeast,1,3-6 implying the possibility
that Jmj protein is a transcription factor. The human jmj gene
has also been identified and maps at 6p24-6p23.7
Interestingly, the mouse jmj gene was independently isolated by
a gene trap method.8
The jmj homozygous mutant (jmjtrap/trap)
mice fail to express normal jmj mRNA.1 The
phenotypes of jmjtrap/trap mice are dependent on
the genetic background.9 The
jmjtrap/trap mice were originally established with
a BALB/cA and 129/Ola mixed genetic background.1 With this
mixed background, approximately half of the
jmjtrap/trap embryos showed neural tube defects,
and all jmjtrap/trap embryos progressively died
between 10.5 days postcoitum (dpc) and 15.5 dpc.1 The
jmjtrap/trap embryos with a BALB/cA, C57BL/6J, or
DBA/2J background, however, displayed no neural tube defects, and
almost all jmjtrap/trap embryos died around 15.5 dpc with hemorrhage and edema.9 In addition, severe
hypoplasia of the fetal liver, thymus, and spleen was observed in
jmjtrap/trap embryos, and in the fetal liver of
jmjtrap/trap embryos with a BALB/cA background, a
decrease in total cell number, an increase in megakaryocytes, and
excessive cell death have been reported.9 On the other
hand, jmj heterozygous mutant (jmj+/trap)
mice showed no apparent abnormalities.1,9 The expression of
jmj gene can be monitored by the  -galactosidase
(-gal) activities in jmj+/trap
embryos.1,4,9 In the fetal liver, the jmj gene is
expressed in megakaryocytes, unidentified hematopoietic cells,
fibroblastic cells, and endothelial cells.9 Because
hypoplasia of the fetal liver in jmjtrap/trap
embryos suggests that hematopoiesis is defective, we further characterized the fetal liver of jmjtrap/trap
embryos with a BALB/cA background. We found abnormalities in the
colony-forming unit-spleen (CFU-S) progenitors in the fetal liver of
jmjtrap/trap embryos. Our studies suggest that the
jmj gene plays a pivotal role in definitive hematopoiesis
during mouse embryogenesis.
| |
MATERIALS AND METHODS |
Mice.
The jmjtrap/trap mice were originally generated
with a 129/Ola and BALB/cA mixed genetic background.1 The
jmjtrap/trap embryos used in this study were
generated by intercross of F16-17 jmj+/trap mice
with a BALB/cA genetic background. The presence of a vaginal plug was
regarded as 0.5 dpc. All embryos were stained with X-gal (Calbiochem-Novabiochem Co, La Jolla, CA), which indicates genotype, and the genotype was confirmed by polymerase chain reaction (PCR) using
genomic DNA isolated from the yolk sacs.1 All embryos that
stained strongly with X-gal were homozygotes. All animals were cared
for by supervised trained technicians in our animal center.
Cytochemical analysis.
To determine the numbers of definitive and primitive erythrocytes in
the peripheral blood, peripheral blood was collected directly from the
umbilical cords of 14.5 dpc and 15.5 dpc embryos through a fine glass
needle. The total number of recovered cells per volume of the
peripheral blood was scored. The ratio of definitive to primitive
erythrocytes was determined by staining peripheral blood smears with
May-Grünwald-Giemsa stain (Merck, Darmstadt, Germany).
The numbers of myeloperoxidase positive (MPO+) cells and
nonspecific esterase positive (NSE+) cells were determined
in single cell preparations from 12.5 dpc fetal livers obtained by
passing a fetal liver sample through into the 26-gauge needle. The
cells were counted, cytocentrifuged, fixed, and stained with
diaminobenzidine (Wako Pure Chemical Co, Osaka, Japan) for
MPO+ or  -naphtylbutyrate (Muto Pure Chemicals Co, Tokyo,
Japan) for NSE+ cells.10 More than 1,000 cells
were counted in each individual sample. The number of positive cells
per fetal liver was estimated from the percentage of positive cells in
the samples.
Histochemical analysis.
For immunohistochemical analysis, the embryos were fixed overnight in
4% paraformaldehyde (Sigma Chemical Co, St Louis, MO) in Dulbecco's
phosphate-buffered saline (PBS, Nissui Pharmaceutical Co, Tokyo, Japan)
at 4°C. The fixative was then replaced in 30% sucrose at 4°C,
embedded in optical cutting temperature (OCT) compound
(Miles Inc, Elkhart, IN) and stored at  80°C. The frozen specimens were cut into 7-µm sections in cryostat, collected on poly-L-lysine-coated slides, and immediately dried. The sections were
stained overnight with biotinylated rat anti-TER-119 antibody (PharMingen Inc, San Diego, CA) or rabbit anti-albumin antibody (Inter-Cell Technologies Inc, Hopewell, NJ) at 4°C. The sections were examined with a Vectastatin ABC Elite kit (Vector Laboratories Inc, Burlingame, CA). The number of TER-119 positive
(TER-119+) or albumin positive (albumin+) cells
per field (magnification × 500) was counted under a microscope.
Flow cytometrical analysis.
Single cell suspensions from 12.5 dpc to 14.5 dpc fetal livers were
prepared as described above. The following monoclonal antibodies were
used: anti-B220/CD45R, anti-c-Kit (ACK2), anti-CD4, anti-CD8,
anti-Gr-1, anti-Thy-1.2, biotinylated anti-c-Kit, biotinylated anti-TER-119, fluorescein isothiocyanate (FITC)-anti-CD45, and FITC-anti-Sca-1 antibodies. ACK2 anti-c-Kit antibody was a kind gift
from Dr S.-I. Nishikawa (Kyoto University, Kyoto, Japan). FITC-anti-CD45 antibody was purchased from Bethesda Research
Laboratories (Gaithersburg, MD). Other antibodies were purchased from
PharMingen Inc. Biotinylated anti-TER-119, ACK2 anti-c-Kit, and
biotinylated anti-c-Kit antibodies were visualized by
FITC-streptavidin (Vector Laboratories Inc), biotinylated antirat IgG
antibody (Vector Laboratories, Inc) followed by FITC-streptavidin and
allophycocyanin (APC)-streptavidin (PharMingen Inc), respectively. The
percentage of TER-119+, CD45 positive (CD45+),
or c-Kit positive (c-Kit+) in 104 fetal liver
cells was determined by FACScan (Becton Dickinson, Mountain View, CA). The number of positive cells per fetal liver was
estimated. The cells negative for lineage markers (anti-B220, anti-CD4,
anti-CD8, anti-Gr-1, and anti-TER-119 antibodies) in the fetal liver
of jmj+/trap embryos at 14.5 dpc were isolated by
using Dynabeads M-450 anti-rat IgG antibody (Dynal, Inc, Lake Success,
NY) and a Dynal MPC-1 magnet (Dynal, Inc). The lineage negative
(Lin) cells were stained with biotinylated
anti-c-Kit and FITC-anti-Sca-1 antibodies. The
Lin, c-Kit+, and Sca-1 positive
(Lin/c-Kit+/Sca-1+) cells
were isolated by FACSvantage (Becton Dickinson) and stained with X-gal.
In vitro colony-forming assay.
Single cell suspensions from the yolk sacs at 10.5 dpc were obtained by
treatment with 0.1% collagenase (Sigma Chemical Co).11 Single cell suspensions from fetal livers were obtained as described above. Cell numbers were determined from trypan blue dye exclusion, and
the cells were suspended in -minimum essential medium
(MEM) (GIBCO Life Technologies Inc, Grand Island, NY)
containing 30% fetal bovine serum (FBS) (JRH BioSciences, Lenexa, KS),
1.2% methylcellulose, 1% deionized bovine serum albumin (BSA), 0.1 mmol/L 2-mercaptoethanol (2-ME), and penicillin-streptomycin. The
methylcellulose, BSA, 2-ME, and penicillin-streptomycin were purchased
from Sigma Chemical Co. Cytokines were added as follows: erythropoietin
(Epo, 2 U/mL; Boehringer Mannheim, Mannheim, Germany) for mature
erythroid progenitors (colony-forming units-erythroid, [CFU-E]),
interleukin-3 (IL-3, 10 ng/mL, Biosource International, Calmario, CA),
stem cell factor (SCF, 50 ng/mL, Biosource International), and Epo for
immature erythroid progenitors (burst-forming units-erythroid,
[BFU-E]), IL-3, SCF, granulocyte-macrophage-colony stimulating factor
(GM-CSF, 10 ng/mL; GIBCO Life Technologies, Inc) for myeloid
progenitors (colony-forming units-granulocyte-macrophage [CFU-GM]).
The cells were cultured in 24-well dishes (Corning Costar Co,
Cambridge, MA), and the numbers of colonies were determined by phase
contrast microscopy after 2 days for CFU-E or 7 days for BFU-E and
CFU-GM. The numbers of colonies per fetal liver were estimated.
Spleen colony-forming assay.
The spleen colony-forming assay was performed as reported
previously.12 Briefly, 5 × 106 fetal
liver cells were suspended in 0.5 mL PBS and injected intravenously into lethally (950 rad) irradiated BALB/cA mice. After 8 days, the
spleens were dissected out, fixed in Bouin's solution (Sigma Chemical
Co), and the number of CFU-S per fetal liver was estimated from the
number of macroscopic colonies.
Reconstitution analysis.
The fetal liver cells obtained from 14.5 dpc
jmj+/trap or jmjtrap/trap
embryos were injected intravenously into lethally (950 rad) irradiated recipient mice (BALB/cA) with 2 × 105 bone marrow
cells of wild-type mice at 6 weeks of age for immediate short-term
radiation protection. Control irradiated mice, which were not
transplanted, died between 10 and 14 days. After 1 month, the bone
marrow, thymus, and spleen of recipient mice were recovered. Biotinylated antibodies, anti-B220, Gr-1, TER-119, and Thy-1.2 (all
purchased from PharMingen Inc) were used for isolation of B lymphoid,
myeloid, erythroid, and T-lymphoid cells, respectively. B220 positive
(B220+) cells from spleen, Gr-1 positive
(Gr-1+), or TER-119+ cells from bone marrow,
and Thy-1.2 positive (Thy-1+) cells from thymus were sorted
by using Dynabeads M-280 streptavidin (Dynal Inc) and a Dynal MPC-E-1
magnet (Dynal Inc). The genomic DNA was recovered from 2 × 106 cells, and the presence of donor-derived hematopoietic
cells was determined by 30 cycles of PCR specific for
jmjtrap allele.1
| |
RESULTS |
The number of definitive erythrocytes is remarkably reduced in the
peripheral blood of jumonji mutant embryos.
We previously reported hypoplasia of the fetal liver, thymus, spleen,
and embryonic lethality around 15.5 dpc in
jmjtrap/trap embryos with a BALB/cA genetic
background,9 suggesting that hematopoiesis is impaired in
jmjtrap/trap embryos. To clarify the defect in
hematopoiesis in jmjtrap/trap embryos, we first
examined the number of erythrocytes in the peripheral blood
(Fig 1). In the peripheral
blood from control littermates, the majority of cells, approximately
80%, is enucleated erythrocytes derived from definitive hematopoiesis
in the fetal liver; in addition, a small number of nucleated, yolk
sac-derived, primitive erythrocytes is observed at 14.5 dpc (Fig 1A).
In contrast, only a few, approximately 17%, enucleated definitive
erythrocytes were seen in the peripheral blood of
jmjtrap/trap embryos at 14.5 dpc (Fig 1B). In
jmjtrap/trap embryos at 14.5 dpc and 15.5 dpc, the
numbers of circulated definitive erythrocytes were 5% to 10% the
level in control (Fig 1C). On the other hand, the number of primitive
erythrocytes showed no significant decrease compared with controls (Fig
1C). The numbers of total erythrocytes in the peripheral blood of
jmjtrap/trap embryos at 14.5 dpc and 15.5 dpc were
15% to 20% of controls (Fig 1C). These data strongly suggest that
definitive, but not primitive, erythropoiesis is severely impaired in
jmjtrap/trap embryos, and embryonic lethality of
jmjtrap/trap embryos seems to be caused by anemia.
View larger version (35K):
[in this window]
[in a new window]
| Fig 1.
The marked reduction in the number of
definitive erythrocytes in the peripheral blood of
jmjtrap/trap embryos. Representative photographs of
erythroid cells from the peripheral blood of control
(jmj+/+) embryos (A) and
jmjtrap/trap embryos (B) at 14.5 dpc are shown. D
and P indicate a definitive erythrocyte and a primitive erythrocyte,
respectively. Scale bar, 50 µm. (C) The numbers of erythrocytes in
the peripheral blood of 14.5 dpc and 15.5 dpc embryos (genotypes shown
below). eryD and eryP indicate definitive erythrocytes and primitive
erythrocytes, respectively.
|
|
The erythroid cells are impaired in the fetal liver of jumonji
mutant embryos.
Definitive erythropoiesis in the fetal liver of
jmjtrap/trap embryos is suggested to be impaired as
shown above, and hypoplasia of the fetal liver occurs in
jmjtrap/trap embryos.9 To know whether
the erythropoiesis is impaired in the fetal liver of
jmjtrap/trap embryos, we stained the sections from
the fetal livers with anti-TER-119 antibody, a specific marker of late
erythroid cells.13 In control littermates, numerous
TER-119+ cells were observed in the fetal livers at 12.5 dpc (Fig 2A). However, in
the fetal liver of jmjtrap/trap embryos at 12.5 dpc, the number of TER-119+ cells was markedly reduced (Fig
2B). The number of TER-119+ cells in the fetal liver was
counted per field and that of jmjtrap/trap embryos
was 19.6% of controls. We next compared the numbers of nonhematopoietic cells, hepatocytes, in the fetal liver of
jmjtrap/trap embryos and those of controls using an
anti-albumin antibody, a marker of hepatocytes. It was important to
determine whether the abnormality in the fetal liver of
jmjtrap/trap embryos occurs only in erythroid cells
or not. In the fetal liver of
jmjtrap/trap embryos, the number of
albumin+ cells was roughly comparable to control embryos
(Fig 2C and D). The number of albumin+ cells in the fetal
liver of jmjtrap/trap embryos per field was 84.8%
of controls. These data suggest that the number of erythroid cells, but
not hepatocytes, is preferentially reduced in the fetal liver of
jmjtrap/trap embryos.
View larger version (158K):
[in this window]
[in a new window]
| Fig 2.
A few late erythroid cells, but not hepatocytes, in the
fetal liver of jmjtrap/trap embryos at 12.5 dpc.
Representative photographs of transverse sections from the fetal liver
of 12.5 dpc jmj+/+ embryos (A and C) and
jmjtrap/trap embryos (B and D) are shown. The
sections were stained with TER-119 antibody (A and B), or albumin
antibody (C and D). The positive cells stain brown. Scale bar, 50 µm.
|
|
View larger version (160K):
[in this window]
[in a new window]
| Fig 8.
No expression of jmj gene in
Lin/c-Kit+/Sca-1+ cells in
the fetal liver. The fetal liver cells of
jmj+/trap embryos at 14.5 dpc were used for
sorting of Lin/c-Kit+/Sca-1+
cells. The sorting gate was shown (A). The
Lin/c-Kit+/Sca-1+ cells were
stained with May-Grünwald-Giemsa (B) and X-gal (C). The X-gal
staining of unfractionated fetal liver cells (D). The arrow indicates a
-gal+ cell. Scale bar, 20 µm.
|
|
The numbers of differentiated erythroid, myeloid, and immature
hematopoietic cells are less in the fetal liver of jumonji
mutant embryos.
To know which lineages of hematopoietic cells are impaired in the fetal
liver of jmjtrap/trap embryos, we investigated the
number of cells representative of hematopoietic lineage,
TER-119+ late erythroid cells, CD45+
leukocytes,14 and c-Kit+ immature hematopoietic
cells.15,16 The numbers of TER-119+,
CD45+, or c-Kit+ cells in the fetal liver of
jmjtrap/trap embryos were lower than in control
embryos at 12.5 dpc to 14.5 dpc (Fig 3A-C).
The numbers of TER-119+ cells per 104 fetal
liver cells in the jmjtrap/trap embryos were lower
than in controls at 12.5 dpc, but comparable to those in controls at
13.5 dpc and 14.5 dpc.
View larger version (18K):
[in this window]
[in a new window]
| Fig 3.
The lower numbers of representative hematopoietic cells
in the fetal livers of jmjtrap/trap embryos. The
numbers of TER-119+ cells (A), CD45+ cells
(B), and c-Kit+ cells (C) per fetal liver from 12.5 dpc
to 14.5 dpc were estimated by flow cytometry. The numbers of
MPO+ cells (D) and NSE+ cells (E) per fetal
liver at 12.5 dpc were estimated by cytochemical analyses. (A through
E) The open columns and circles indicate the average numbers of
positive cells from jmj+/+ and
jmj+/trap embryos, and the closed columns and
circles indicate the average numbers from
jmjtrap/trap embryos. In all cases, more than three
embryos were analyzed. The error bars indicate SEM.
|
|
CD45+ leukocytes include myeloid and lymphoid cells. The
differentiated lymphoid cells represented as B220+ B
lymphocytes or Thy-1 strongly positive T lymphocytes are undetectable in the fetal liver before 15.5 dpc.13 Therefore, we could
not examine the number of lymphocytes. To assess the number of myeloid cells, we analyzed the number of MPO+ or NSE+
cells in the fetal liver of jmjtrap/trap embryos at
12.5 dpc by cytochemical analysis. The activities of MPO and NSE have
been reported to be restricted in myeloid lineage cells.17
The number of MPO+ or NSE+ cells in the fetal
liver of jmjtrap/trap embryos at 12.5 dpc was 16%
or 46% of controls, respectively (Fig 3D and E). In megakaryocytic
lineage cells in the fetal liver of jmjtrap/trap
embryos, we previously showed an increased percentage of
megakaryocytes.9 Taken together, the numbers of
differentiated erythroid and myeloid cells, but not megakaryocytes,
were lower in the fetal liver of jmjtrap/trap
embryos.
The numbers of erythroid and myeloid progenitors of definitive
hematopoiesis are low in the fetal liver of jumonji mutant
embryos.
We next investigated the number of erythroid and myeloid-committed
progenitors in the fetal liver of jmjtrap/trap
embryos using an in vitro colony-forming assay to know why the numbers
of differentiated erythroid and myeloid cells were less. The numbers of
BFU-E, CFU-E, and CFU-GM colonies from the fetal liver of
jmjtrap/trap embryos were fewer compared with
controls at all stages examined (Fig 4).
Moreover, the numbers of BFU-E in the fetal livers, which gradually
increase in control mice, showed no increase in
jmjtrap/trap embryos between 12.5 dpc and 14.5 dpc
(Fig 4A). The number of CFU-GM in the fetal livers between 12.5 dpc and
14.5 dpc also increased significantly in controls, but not in
jmjtrap/trap embryos (Fig 4C). However, the gross
morphologies of the erythroid and myeloid colonies from
jmjtrap/trap embryos were indistinguishable from
those of controls (data not shown). These results strongly suggest that
the lower numbers of differentiated erythroid and myeloid cells in the
fetal livers of jmjtrap/trap embryos are caused by
the reduction in the numbers of erythroid and myeloid progenitors.
View larger version (20K):
[in this window]
[in a new window]
| Fig 4.
The reduction in the numbers of hematopoietic progenitors
in the fetal livers of jmjtrap/trap embryos. The
numbers per fetal liver of BFU-E (A) and CFU-E colonies (B) in 11.5 dpc
to 14.5 dpc embryos and CFU-GM colonies (C) in 12.5 dpc and 14.5 dpc
embryos were determined. The open and closed columns indicate the
average numbers of colonies from jmj+/+ and
jmj+/trap embryos and from
jmjtrap/trap embryos, respectively. The numbers of
fetal livers analyzed are shown above the columns and the error bars
indicate SEM.
|
|
Erythroid and myeloid progenitors of definitive hematopoiesis in the
yolk sac are unaffected in jumonji mutant embryos.
To understand why the numbers of BFU-E and CFU-GM progenitors in the
fetal liver of jmjtrap/trap embryos were less, we
first examined the numbers of BFU-E and CFU-GM in the yolk sacs at 10.5 dpc. Erythroid and myeloid progenitors of definitive hematopoiesis in
the fetal liver of normal embryos seem to be derived from the
colonization from the yolk sac18 and from the
differentiation from hematopoietic stem cells (HSCs) in the fetal
liver. The numbers of BFU-E and CFU-GM colonies from the
yolk sac of jmjtrap/trap embryos were equivalent to
those from controls at 10.5 dpc (Fig 5),
suggesting the possibility that the numbers of erythroid and myeloid
progenitors of definitive hematopoiesis in
jmjtrap/trap embryos are the same as in control
embryos before colonization to the fetal liver.
View larger version (20K):
[in this window]
[in a new window]
| Fig 5.
Hematopoietic progenitors of definitive hematopoiesis in
10.5 dpc yolk sacs are unaffected in jmjtrap/trap
embryos. (A) The number of BFU-E colonies per yolk sac.
jmj+/+ and jmj+/trap
embryos (n = 6) and jmjtrap/trap
embryos (n = 6) were analyzed. (B) The number of CFU-GM colonies per
yolk sac. jmj+/+ and
jmj+/trap embryos (n = 4) and
jmjtrap/trap embryos (n = 4) were analyzed. The
open and closed circles represent the average numbers of colonies from
jmj+/+ and jmj+/trap
embryos and from jmjtrap/trap embryos,
respectively. The error bars indicate SEM.
|
|
The number of CFU-S progenitors is significantly reduced in the fetal
liver of jumonji mutant embryos.
We next examined the number of CFU-S, erythroid and myeloid common
progenitors to know why the numbers of erythroid and myeloid progenitors in the fetal liver of jmjtrap/trap
embryos were less and failed to increase. When 5 × 106 cells from 13.5 dpc fetal livers were used for single
injection, numerous colonies were observed from controls, but only a
few colonies from jmjtrap/trap embryos
(Fig 6A and B). As shown in Fig 6C, the
average number ± standard error of mean (SEM) of CFU-S per fetal
liver from control and jmjtrap/trap embryos at 13.5 dpc was 31.6 ± 3.6 (n = 9) and 1.0 ± 0.5 (n = 5), respectively
(P < .001). In addition, the average number of CFU-S per
106 fetal liver cells of control and
jmjtrap/trap embryos at 13.5 dpc was 5.0 and 0.4, respectively. These results strongly suggest that the decrease in the
numbers of erythroid and myeloid progenitors in the fetal liver of
jmjtrap/trap embryos is caused by the lower number
of CFU-S progenitors.
View larger version (38K):
[in this window]
[in a new window]
| Fig 6.
The significant reduction in the number of CFU-S
progenitors in the fetal livers of jmjtrap/trap
embryos. Representative photographs of CFU-S colonies from 5 × 106 cells from 13.5 dpc fetal livers of
jmj+/+ (A) and jmjtrap/trap
embryos (B). Scale bar, 2 mm. (C) Number of CFU-S colonies per fetal
liver. The open and closed columns indicate the average numbers of
colonies from jmj+/+ and
jmj+/trap embryos and from
jmjtrap/trap embryos, respectively. The number of
experiments is shown above each column and the error bars indicate
SEM.
|
|
The HSCs in the fetal liver of jumonji mutant embryos can
reconstitute the hematopoietic system of lethally irradiated recipient
mice.
To know whether the hematopoietic defect of
jmjtrap/trap embryos is caused cell-autonomously or
not, we examined reconstitution analysis using fetal liver cells. The
strategy is shown in Fig 7A. Lethally irradiated recipients were transplanted with 4 × 106
or 7 × 106 cells from 14.5 dpc fetal livers of
jmj+/trap embryos (0.4 or 0.7 embryo equivalent,
respectively) or those of jmjtrap/trap embryos (1.2 or 2.1 embryo equivalent). After 1 month, fetal liver cells from
jmjtrap/trap embryos reconstituted hematopoietic
cells in the bone marrow, thymus, and spleen of the recipients, as well
as those from jmj+/trap embryos, when 7 × 106 cells were injected (Fig 7B and C). Moreover,
donor-derived hematopoietic cells were detected in T lymphoid
(Thy-1+ cells in thymus), B lymphoid (B220+
cells in spleen), erythroid (TER-119+ cells in bone
marrow), and myeloid (Gr-1+ cells in bone marrow) lineage
cells of recipients transplanted with the fetal liver cells from
jmj+/trap or jmjtrap/trap
embryos (Fig 7D and E). PCR products specific for
jmjtrap allele were not detected in wild-type mice
(data not shown). However, PCR products specific for
jmjtrap/trap allele from the recipients
transplanted with 4 × 106 fetal liver cells in
jmjtrap/trap embryos showed weak signals compared
with those in jmj+/trap embryos (Fig 7B and C). On
the other hand, PCR products of wild-type jmj allele in
recipients transplanted with fetal liver cells of jmjtrap/trap embryos was equivalent to those in
controls (Fig 7F through I). Although these PCR analyses were
semiquantitative, these results suggest that the number of HSCs may be
reduced in the fetal liver of jmjtrap/trap embryos,
but HSCs in the fetal liver of jmjtrap/trap embryos
have a potential for differentiation into representative hematopoietic
cell lineages.
View larger version (52K):
[in this window]
[in a new window]
| Fig 7.
Multilineage reconstitution of recipient mice
transplanted with the fetal liver cells of
jmjtrap/trap embryos. Strategy of reconstitution
analysis (A). Lethally irradiated recipient mice were injected with the
fetal liver cells of jmjtrap/trap and
jmj+/trap and after 1 month, the presence of
donor-derived cells was determined by PCR. The representative results
of genotype determined by PCR analysis specific for
jmjtrap allele (B-E) and wild-type jmj
allele (F through I). The recipients transplanted with the fetal liver
cells of jmj+/trap embryos (B, D, F, and H) and
jmjtrap/trap embryos (C, E, G, and I) were
examined. BM, SP, and TH indicate bone marrow, spleen, and thymus,
respectively. TER, Gr, B220, and Thy indicate TER+,
Gr-1+, B220+, and Thy-1+
cells, respectively. TER+ and Gr-1+ cells
were isolated from the bone marrow. B220+ and
Thy-1+ cells were from spleen and thymus, respectively.
|
|
No expression of jumonji gene in
Lin/c-Kit+/Sca-1+ cells in
the fetal liver.
It is important to know whether the jmj gene is expressed in
HSCs or not. In the fetal liver, we previously showed that the jmj gene is expressed in megakaryocytes, small unidentified
cells, fibroblastic cells, and endothelial cells.9 In
addition, the expression of jmj gene was also detected in a few
CD34+ cells and a subset of GATA-1+ cells, but
not in any TER-119+ cells and albumin+
hepatocytes in the fetal liver (Kitajima et al, in
preparation). The HSC activity is reported to be found in
Lin/c-Kit+/Sca-1+ cells in
the fetal liver.19
Lin/c-Kit+/Sca-1+ cells in
the fetal liver of jmj+/trap embryos at 14.5 dpc
were then sorted (Fig 8A [see page 90]), and the expression of
jmj gene in the
Lin/c-Kit+/Sca-1+ cells was
examined by X-gal staining, because the -gal activities mimic
faithfully the expression of jmj gene in
jmj+/trap embryos.1
Lin/c-Kit+/Sca-1+ cells
(total of 197 cells) were examined under a microscope. In the
Lin/c-Kit+/Sca-1+ cells
sorted, all observed cells were blastic cells (Fig 8B). However, in
Lin/c-Kit+/Sca-1+ cells of
jmj+/trap embryos, no -gal activity was detected
(Fig 8C). On the other hand, in unfractionated cells in the fetal
liver, small hematopoietic cells were stained with X-gal (Fig 8D), as
previously described.9 Thus, the jmj gene is not
expressed in the hematopoietic stem cells in the fetal liver.
| |
DISCUSSION |
The reduction in the number of definitive erythrocytes in the
peripheral blood of jumonji mutant embryos.
Fetal liver-derived erythrocytes begin to circulate in the peripheral
blood at about 12.5 dpc and completely replace the primitive erythrocytes by 16.5 dpc. In the peripheral blood of
jmjtrap/trap embryos, the numbers of definitive,
but not primitive, erythrocytes were 90% to 95% lower than in
controls at 14.5 dpc and 15.5 dpc (Fig 1C). This result strongly
suggests that definitive, but not primitive, hematopoiesis is severely
impaired in jmjtrap/trap embryos.
In the peripheral blood of jmjtrap/trap embryos,
the numbers of total erythrocytes were 15% to 20% of those in
controls (Fig 1C). This result strongly suggests that embryonic
lethality of jmjtrap/trap embryos is caused by
anemia. The reduction in the numbers of circulated definitive
erythrocytes is also reported in proto-oncogene c-myb or
homeobox gene Hlx null mice.20,21 In c-myb
null embryos, hematocrit of peripheral blood was approximately 15% of
controls between 14.5 dpc and 15.5 dpc.20 In Hlx
null embryos, the number of total erythrocytes in the peripheral blood
was approximately 30% of that in control embryos at 14.5 dpc.21 These mutant mice die at about 15.5 dpc probably due
to anemia.20,21 These studies support the notion that the
embryonic mortality of jmjtrap/trap embryos around
15.5 dpc is due to anemia.
The defects in definitive hematopoiesis in the fetal liver of
jumonji mutant embryos.
In the fetal liver of jmjtrap/trap embryos, the
numbers of TER-119+ late erythroid cells, CD45+
leukocytes, c-Kit+ immature hematopoietic cells, and
MPO+ or NSE+ myeloid cells were lower (Fig 3).
On the other hand, hepatocytes appear not to be severely affected (Fig
2C and D). It is suggested that hypoplasia of the fetal liver is caused
by the reduction in the number of TER-119+ cells, because
the vast majority of cells in the fetal liver after 13.5 dpc is
erythroid lineage.15 The reduction in the numbers of
differentiated erythroid and myeloid cells in the fetal liver of
jmjtrap/trap embryos raises a question concerning
the ability of erythroid and myeloid progenitors in
jmjtrap/trap embryos to differentiate into mature
cells. Although the numbers of differentiated erythroid and myeloid
cells were lower, these cells were present in the fetal liver and
peripheral blood of jmjtrap/trap embryos (Figs 1
and 3), suggesting that erythroid and myeloid progenitors of
jmjtrap/trap embryos can pursue terminal
differentiation.
In the fetal liver of jmjtrap/trap embryos, the
number of BFU-E and CFU-GM failed to increase as in controls (Fig 4A
and C). The significant reduction in the number of CFU-S in the fetal
liver of jmjtrap/trap embryos (Fig 6C) suggests
that the failure of BFU-E and CFU-GM progenitors is caused by a fewer
supply of BFU-E and CFU-GM progenitors from CFU-S progenitors. The
presence of BFU-E and CFU-GM in the fetal liver of
jmjtrap/trap embryos in the early stages of
definitive hematopoiesis (11.5 dpc and 12.5 dpc, Fig 4A and C) can be
explained by the colonization of erythroid and myeloid progenitors from
the yolk sac.18
The function of the jumonji gene in definitive hematopoiesis.
In the fetal liver of jmjtrap/trap embryos at 13.5 dpc, the number of CFU-S progenitors was less than 5% that in controls
(Fig 6). This result strongly suggests that hypoplasia of the fetal
liver and embryonic lethality are caused by the significant reduction in the number of CFU-S progenitors in the fetal liver of
jmjtrap/trap embryos. On the other hand, in the
fetal liver of jmjtrap/trap embryos, in vitro
development of erythroid and myeloid progenitors are normal (data not
shown), the HSCs reconstitute all hematopoietic lineages in irradiated
recipients (Fig 7), and
Lin/c-Kit+/Sca-1+ cells,
known to have HSC activity, do not express the jmj gene (Fig
8). These results suggest that the reduced number of CFU-S progenitors
in the fetal liver of jmjtrap/trap embryos is
caused by an environmental defect.
The generations of CFU-S progenitors and long-term repopulating
(LTR)-HSCs are reported to occur in the aorta-gonad-mesonephros (AGM)
region at about 10.5 to 11.5 dpc.22-27 Moreover, the CFU-S progenitors and LTR-HSCs in the AGM region seem to migrate and colonize
the fetal liver.25,26 The results of CFU-S, reconstitution, and expression analyses (Figs 6 through 8) suggest an impairment of the
generation of CFU-S in the AGM region, colonization of CFU-S
progenitors from the AGM region to the fetal liver, or hematopoietic environment, which supports expansion of CFU-S progenitors in the fetal
liver.
First, the generation of HSCs and CFU-S progenitors in the AGM region
of jmjtrap/trap embryos and the expression of
jmj gene in the AGM region remains to be further examined.
Recently, it was reported that immature hematopoietic cells including
multipotent hematopoietic progenitors can be emerged by in vitro
culture of AGM cells.28 We examined the generation of
hematopoietic cells in the AGM region by this culture system. In the
culture of AGM cells of jmjtrap/trap embryos,
immature hematopoietic cells were normally developed (K.K., unpublished
data, June 1997). This in vitro culture system suggests
that the AGM region of jmjtrap/trap embryos has a
potential for the generation of hematopoietic cells. Next, in
jmjtrap/trap embryos, the fetal liver environment
supporting hematopoiesis may not be severely impaired, because the
number of CFU-S in the fetal liver of jmjtrap/trap
embryos at 14.5 dpc increased to approximately sevenfold of that at
13.5 dpc (Fig 6). Finally, there is a possibility that the colonization
and migration of CFU-S progenitors from the AGM region to the fetal
liver may be affected by the defect of nonhematopoietic cells such as
endothelial cells in the fetal liver and dorsal aorta of
jmjtrap/trap embryos. The morphologic analysis of
endothelial cells in jmjtrap/trap embryos is
currently in progress.
In summary, our results suggest that the jmj gene has an
important role in the hematopoietic microenvironment. Further
investigation of the jmj gene will contribute to our
understanding of the development of the hematopoietic system in
mammals.
| |
ACKNOWLEDGMENT |
The authors thank Dr Shin-Ichi Nishikawa (Kyoto University, Japan) for
the gift of ACK2 antibody, Dr Hajime Takayama and Yoshiko Shirota
(Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan) for
supporting FACS analysis, CFU-S assay, and reconstitution experiments,
Takashi Sekiguchi (Tokyo University, Japan) for cell sorting analysis,
and Dr Toru Nakano (Osaka University, Japan) for discussion and
critical review of the manuscript.
| |
FOOTNOTES |
Submitted April 21, 1998;
accepted August 24, 1998.
Supported in part by a Grant-in-Aid for Creative Basic Research from
the Ministry of Education, Science, Sports, and Culture of Japan.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Takashi Takeuchi, PhD,
Mitsubishi Kasei Institute of Life Sciences, 11, Minamiooya, Machida,
Tokyo 194-8511, Japan; e-mail: take{at}libra.ls.m-kagaku.co.jp.
| |
REFERENCES |
1.
Takeuchi T, Yamazaki Y, Katoh-Fukui Y, Tsuchiya R, Kondo S, Motoyama J, Higashinakagawa T:
Gene trap capture of a novel mouse gene, jumonji, required for neural tube formation.
Genes Dev
9:1211, 1995[Abstract/Free Full Text]
2.
Takeuchi T:
jumonji gene maps to mouse chromosome 13.
Genomics
45:240, 1997[Medline]
[Order article via Infotrieve]
3.
Gregory SL, Kortschak RD, Kalionis B, Saint R:
Characterization of the dead ringer gene identifies a novel, highly conserved family of sequence-specific DNA-binding proteins.
Mol Cell Biol
16:792, 1996[Abstract]
4.
Takeuchi T:
A gene trap approach to identify genes that control development.
Dev Growth Differ
39:127, 1997[Medline]
[Order article via Infotrieve]
5.
Herrscher RF, Kaplan MH, Lelsz DL, Das C, Scheuermann R, Tucker PW:
The immunoglobulin heavy-chain matrix-associating regions are bound by Bright: A B cell-specific trans-activator that describes a new DNA-binding protein family.
Genes Dev
9:3067, 1995[Abstract/Free Full Text]
6.
Peterson CL, Herskowitz I:
Characterization of the yeast SWI1, SWI2, and SWI3 genes, which encode a global activator of transcription.
Cell
68:573, 1992[Medline]
[Order article via Infotrieve]
7.
Bergé-Lefranc JL, Jay P, Massacrier A, Cau P, Mattei MG, Bauer S, Marsollier C, Berta P, Fontes M:
Characterization of the human jumonji gene.
Hum Mol Genet
5:1637, 1996[Abstract/Free Full Text]
8.
Baker RK, Haendel MA, Swanson BJ, Shambaugh JC, Micales BK, Lyons GE:
In vitro preselection of gene-trapped embryonic stem cell clones for characterizing novel developmentally regulated genes in the mouse.
Dev Biol
185:201, 1997[Medline]
[Order article via Infotrieve]
9.
Motoyama J, Kitajima K, Kojima M, Kondo S, Takeuchi T:
Organogenesis of the liver, thymus and spleen is affected in jumonji mutant mice.
Mech Dev
66:27, 1997[Medline]
[Order article via Infotrieve]
10.
Shibata A, Bennett JM, Castoldi GL, Catovsky D, Flandrin G, Jaffe ES, Katayama I, Nanba K, Schmalzl F, Yam LT, Lewis SM:
Recommended methods for cytological procedures in haematology. International Committee for Standardization in Haematology (ICSH).
Clin Lab Haematol
7:55, 1985[Medline]
[Order article via Infotrieve]
11.
Wong PMC, Chung SW, Chui DHK, Eaves CJ:
Properties of the earliest clonogenic hemopoietic precursors to appear in the developing murine yolk sac.
Proc Natl Acad Sci USA
83:3851, 1986[Abstract/Free Full Text]
12.
Till JE, McCulloch EA:
A direct measurement of the radiation sensitivity of normal mouse bone marrow cells.
Radiat Res
14:213, 1961[Medline]
[Order article via Infotrieve]
13.
Ikuta K, Kina T, MacNeil I, Uchida N, Peault B, Chien YH, Weissman IL:
A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells.
Cell
62:863, 1990[Medline]
[Order article via Infotrieve]
14.
Springer T, Galfrè G, Secher DS, Milstein C:
Monoclonal xenogeneic antibodies to murine cell surface antigens: Identification of novel leukocyte differentiation antigens.
Eur J Immunol
8:539, 1978[Medline]
[Order article via Infotrieve]
15.
Ogawa M, Nishikawa S, Yoshinaga K, Hayashi S, Kunisada T, Nakao J, Kina T, Sudo T, Kodama H, Nishikawa S:
Expression and function of c-Kit in fetal hemopoietic progenitor cells: Transition from the early c-Kit-independent to the late c-Kit-dependent wave of hemopoiesis in the murine embryo.
Development
117:1089, 1993[Abstract]
16.
Ogawa M, Matsuzaki Y, Nishikawa S, Hayashi S, Kunisada T, Sudo T, Kina T, Nakauchi H, Nishikawa S:
Expression and function of c-kit in hemopoietic progenitor cells.
J Exp Med
174:63, 1991[Abstract/Free Full Text]
17.
Yam LT, Li CY, Crosby WH:
Cytochemical identification of monocytes and granulocytes.
Am J Clin Pathol
55:283, 1971[Medline]
[Order article via Infotrieve]
18.
Cudennec CA, Thiery JP, Le Douarin NM:
In vitro induction of adult erythropoiesis in early mouse yolk sac.
Proc Natl Acad Sci USA
78:2412, 1981[Abstract/Free Full Text]
19.
Randall TD, Lund FE, Howard MC, Weissman IL:
Expression of murine CD38 defines a population of long-term reconstituting hematopoietic stem cells.
Blood
87:4057, 1996[Abstract/Free Full Text]
20.
Mucenski ML, McLain K, Kier AB, Swerdlow SH, Schreiner C M, Miller TA, Pietryga DW, Scott W J Jr, Potter SS:
A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis.
Cell
65:677, 1991[Medline]
[Order article via Infotrieve]
21.
Hentsch B, Lyons I, Li R, Hartley L, Lints TJ, Adams JM, Harvey RP:
Hlx homeo box gene is essential for an inductive tissue interaction that drives expansion of embryonic liver and gut.
Genes Dev
10:70, 1996[Abstract/Free Full Text]
22.
Dzierzak E, Medvinsky A:
Mouse embryonic hematopoiesis.
Trends Genet
11:359, 1995[Medline]
[Order article via Infotrieve]
23.
Medvinsky AL, Samoylina NL, Müller AM, Dzierzak EA:
An early pre-liver intraembryonic source of CFU-S in the developing mouse.
Nature
364:64, 1993[Medline]
[Order article via Infotrieve]
24.
Müller AM, Medvinsky A, Strouboulis J, Grosveld F, Dzierzak E:
Development of hematopoietic stem cell activity in the mouse embryo.
Immunity
1:291, 1994[Medline]
[Order article via Infotrieve]
25.
Medvinsky AL, Gan OI, Semenova ML, Samoylina NL:
Development of day-8 colony-forming unit-spleen hematopoietic progenitors during early murine embryogenesis: Spatial and temporal mapping.
Blood
87:557, 1996[Abstract/Free Full Text]
26.
Medvinsky A, Dzierzak E:
Definitive hematopoiesis is autonomously initiated by the AGM region.
Cell
86:897, 1996[Medline]
[Order article via Infotrieve]
27.
Sánchez MJ, Holmes A, Miles C, Dzierzak E:
Characterization of the first definitive hematopoietic stem cells in the AGM and liver of the mouse embryo.
Immunity
5:513, 1996[Medline]
[Order article via Infotrieve]
28.
Mukouyama Y, Hara T, Xu M, Tamura K, Donovan PJ, Kim H, Kogo H, Tsuji K, Nakahata T, Miyajima A:
In vitro expansion of murine multipotential hematopoietic progenitors from the embryonic aorta-gonad-mesonephros region.
Immunity
8:105, 1998[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
R. M. O'Connell, D. S. Rao, A. A. Chaudhuri, M. P. Boldin, K. D. Taganov, J. Nicoll, R. L. Paquette, and D. Baltimore
Sustained expression of microRNA-155 in hematopoietic stem cells causes a myeloproliferative disorder
J. Exp. Med.,
March 17, 2008;
205(3):
585 - 594.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Chateauvieux, J.-L. Ichante, B. Delorme, V. Frouin, G. Pietu, A. Langonne, N. Gallay, L. Sensebe, M. T. Martin, K. A. Moore, et al.
Molecular profile of mouse stromal mesenchymal stem cells
Physiol Genomics,
April 24, 2007;
29(2):
128 - 138.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T.-g. Kim, J. Chen, J. Sadoshima, and Y. Lee
Jumonji Represses Atrial Natriuretic Factor Gene Expression by Inhibiting Transcriptional Activities of Cardiac Transcription Factors
Mol. Cell. Biol.,
December 1, 2004;
24(23):
10151 - 10160.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Chagraoui, A. Lepage-Noll, A. Anjo, G. Uzan, and P. Charbord
Fetal liver stroma consists of cells in epithelial-to-mesenchymal transition
Blood,
April 15, 2003;
101(8):
2973 - 2982.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. D. Car and V. M. Eng
Special Considerations in the Evaluation of the Hematology and Hemostasis of Mutant Mice
Vet. Pathol.,
January 1, 2001;
38(1):
20 - 30.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Lee, A. J. Song, R. Baker, B. Micales, S. J. Conway, and G. E. Lyons
Jumonji, a Nuclear Protein That Is Necessary for Normal Heart Development
Circ. Res.,
May 12, 2000;
86(9):
932 - 938.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction