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Prepublished online as a Blood First Edition Paper on July 12, 2002; DOI 10.1182/blood-2002-02-0398.
 Previous Article | Table of Contents | Next Article 
Blood, 1 January 2003, Vol. 101, No. 1, pp. 124-133
HEMATOPOIESIS
Disruption of Smad5 gene leads to enhanced
proliferation of high-proliferative potential precursors during
embryonic hematopoiesis
Bing Liu,
Yanxun Sun,
Feizi Jiang,
Shuangxi Zhang,
Ying Wu,
Yu Lan,
Xiao Yang, and
Ning Mao
From the Department of Cell Biology, Institute of Basic
Medical Sciences, Genetic Laboratory of Development and Diseases,
Institute of Biotechnology, and Department of Hematology, Yan Jing
Hospital, Beijing, Peoples' Republic of China.
 |
Abstract |
SMAD proteins are downstream signal transducers of the
transforming growth factor  (TGF-) superfamily, which serve as
pleiotropic regulators in embryonic and adult hematopoiesis. SMAD5,
initially considered to mediate bone morphogenetic proteins
(BMPs) signals, can also transduce the inhibitory signal of
TGF-1 on proliferation of hematopoietic progenitors derived
from human bone marrow. To define its specific role in regulation of
primitive multipotential progenitors during early embryonic
hematopoiesis, we examined Smad5 / yolk sacs at E9.0 to
9.5 and detected an elevated number of high-proliferative potential
colony-forming cells (HPP-CFCs) with enhanced replating potential. To
exclude the possible influence of microenvironmental deficit on
embryonic hematopoiesis in vivo, we performed in vitro embryonic stem
(ES) cell differentiation assay and investigated the HPP-CFCs in
particular. Smad5/ embryoid bodies (EBs) contained an
elevated number of blast colony-forming cells (BL-CFCs), the in vitro
equivalent of hemangioblast, in contrast to reduced proliferation of
primitive erythroid precursors (Ery/Ps) within the mutant EBs. More
importantly, profoundly increased frequency of HPP-CFCs, featured with
a gene-dosage effect, was detected within day 6 Smad5/
EBs compared with the wild type. In addition, Smad5/
HPP-CFCs displayed enhanced self-renewal capacity and decreased sensitivity to TGF-1 inhibition, suggesting a critical role of Smad5
in TGF-1 regulation of embryonic HPP-CFCs. Consistently, reverse
transcription-polymerase chain reaction analysis detected alterations
of the transcription factors including GATA-2 and AML1 as well as
cytokine receptors in Smad5/ HPP-CFC colonies.
Together, these data define an important function of SMAD5 in negative
regulation of high-proliferative potential precursors during
embryonic hematopoiesis.
(Blood. 2003;101:124-133)
© 2003 by The American Society of Hematology.
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Introduction |
The transforming growth factor (TGF-)
superfamily, a large group of highly conserved growth factors including
TGF-, activins, and bone morphogenetic proteins (BMPs),
regulate a wide variety of cellular functions such as proliferation,
differentiation, apoptosis, and migration. Signals from these growth
factors are transduced by a group of SMAD
proteins.1,2 To date, there are 9 vertebrate SMADs,
including the receptor-activated pathway-specific SMADs (R-SMADs),
SMAD1-3, 5, and 8, the common mediator SMAD4 and SMAD4, and the
inhibitory SMADs, SMAD6 and 7. Signaling is initiated when the ligand
induces assembly of a heteromeric complex of type II TGF- receptors
(TRII) and type I TGF- receptors (TRI). Then R-SMADs
are directly phosphorylated by activated TRI. On phosphorylation,
R-SMADs interact with SMAD4 to form heteromeric complexes that
translocate to the nucleus, where they act as transcription factors to
regulate the transcriptional response of the target genes. Original in
vitro studies suggest that SMAD2 and 3 act downstream of the TGF-
and activin receptors, whereas SMAD1 and 5 respond to BMP
signals.3-7
Previous studies have strongly shown that TGF- can either in vivo or
in vitro preferentially inhibit proliferation of the most primitive
types of hematopoietic cells, long-term culture-initiating cells
(LTC-ICs) and their presumed immediate progeny, high-proliferative potential colony-forming cells (HPP-CFCs), but spare more mature low-proliferative potential colony-forming cells
(LPP-CFCs).8-11 Although conventionally thought to respond
to BMP signals, a recent finding demonstrates that SMAD5 is also
capable of transducing the inhibitory signal of TGF-1 and TGF-2
on proliferation of hematopoietic progenitor cells from human adult
bone marrow.12
Whether SMAD5 can also play a role in negative regulation of primitive
multipotential progenitors by TGF- during embryonic hematopoiesis is
particularly investigated in this study. Targeted disruption of the
Smad5 gene results in multiple defects and embryonic lethality at E10.5 to E11.5.13,14 Preliminary analysis has shown that Smad5/ yolk sacs can give rise to an
increased number of granulocyte-macrophage colony-forming units
(CFU-GMs); therefore, it is highly probable that SMAD5 may also mediate
the inhibitory signal of TGF- on proliferation of hematopoietic
progenitor cells during embryonic development. Considering the
selective modulation of TGF- on primitive hematopoietic progenitors
from bone marrow and cord blood, the appropriate choice of the
precursors to be focused may be critical. Previous in vitro assays have
defined the presence of quiescent bone marrow progenitors that
represent cells close to, or actually within, the hematopoietic stem
cell (HSC) compartment. HPP-CFCs, first described as murine bone marrow
cells giving rise to macroscopic colonies and quiescent cells with
relative resistance to 5-fluorouracil treatment, are the earliest
multipotential hematopoietic precursors that can be cultured in vitro
in the absence of stromal cells.15 During embryonic
hematopoiesis, it is detected first at early somite stages (E8.25) in
mouse yolk sac and can be recapitulated using the differentiation model
of embryonic stem (ES) cells.16 Whereas CFU-GMs detected
in 7-day CFC assay represent hematopoietic progenitors at a relatively
mature stage, accordingly, the specific concern for HPP-CFCs is more
relevant in terms of the primitiveness (stemness).
Moreover, because the defective microenvironment, including
angiogenesis defect and mesenchyme apoptosis, is detected in early Smad5/ embryos, direct analysis of the embryonic
hematopoiesis in these mutant embryos would not give the most reliable
results.13 Alternatively, the in vitro hematopoietic
differentiation of ES cells has been shown to recapitulate the early
embryonic hematopoiesis while circumventing the drawbacks of defective
development and early death of some mutant embryos.17
Hence, in this study, we capitalized on this system and analyzed
hematopoietic precursors such as blast colony-forming cells (BL-CFCs),
primitive erythroid precursors (Ery/Ps), and definitive HPP-CFCs,
sequentially initiating within embryoid bodies (EBs), to identify the
roles of SMAD5 in regulation of these cells.
Here we demonstrated increased frequency and self-renewal capacity of
HPP-CFCs from Smad5/ E9.0 to 9.5 yolk sac and ES cells
with the latter characterized by a gene-dosage effect and more
profoundly affected. Moreover, reduced sensitivity of the
mutant HPP-CFC to TGF-1 inhibition further supported the involvement
of the Smad5 gene in the TGF-1/HPP-CFC regulatory
pathway. In contrast to enhanced frequency of BL-CFCs within
Smad5/ EBs, reduced proliferation of Ery/P was
observed. These data may suggest essential and pleiotropic involvements
of SMAD5 and its upstream TGF- superfamily members during
embryonic hematopoiesis.
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Materials and methods |
ES cell lines and culture conditions
Smad5 double-knockout ES cell lines were generated from TC1 ES
cells through homologous recombination and one wild-type, one heterozygous Smad5+/, and one homozygous
Smad5/ ES cell line were used in this study and
maintained as previously described.13,18
ES cells in vitro hematopoietic differentiation
The methods used for the hematopoietic differentiation of ES
cells were essentially as described by Keller et al.17 For the generation of EBs, ES cells (6000 cells/3.5-cm Petri dish) were
plated into methylcellulose medium without extra cytokines and after 7 days of differentiation c-kit ligand (KL; 20 ng/mL) and interleukin 11 (IL-11; 10 ng/mL) were added. At day 12 of ES cell differentiation,
hematopoietic EBs were scored according to a previously described
standard.19
BL-CFC assay
To assess BL-CFCs, blast colonies were generated as described
previously.20 Briefly, blast colonies were generated from dispersed day 3.5 EBs cells in the presence of vascular endothelial growth factor (VEGF; 5 ng/mL) and KL (50 ng/mL) and scored after 4 to 6 days of incubation.
HPP-CFC assay
Disaggregated cells from day 6 EBs as well as E9.0 to 9.5 yolk
sac were replated into methylcellulose HPP-CFC cultures containing KL
(50 ng/mL), IL-3 (20 ng/mL), IL-11 (20 ng/mL), granulocyte-macrophage colony-stimulating factor (GM-CSF; 20 ng/mL), and erythropoietin (Epo;
6 U/mL) in 3.5-cm Petri dishes. Epo and TGF-2 were purchased from
Kirin Brewery (Tokyo, Japan) and R & D Systems
(Minneapolis, MN), respectively, and the other cytokines were
obtained from Peprotech (Rocky Hill, NJ). For HPP-CFC
identification, compact colonies (> 0.5 mm) or more diffuse colonies
(> 1.0 mm) were scored as HPP-CFCs by an inverted microscope after
incubation at 37°C for 2 weeks. Other small colonies, such as CFU-mix
and CFU-GM, were scored after 7 to 10 days of culture. For detection of
Ery/Ps, disaggregated EB cells at the indicated time were cultured in medium containing Epo (6 U/mL) and scored after 5 to 7 days of incubation.
Replating experiments
At 14 days of culture, plucked HPP-CFC colonies were either
individually or bulk resuspended in 200 µL Iscove modified Dulbecco medium (IMDM) to form single-cell suspensions, then replated into HPP-CFC culture. Secondary HPP-CFCs or other more committed colonies were scored after 14 and 7 days of culture. The tertiary cultures were
performed by replating 1 × 105 cells from secondary
HPP-CFC culture into HPP-CFC medium after 2 weeks of incubation. For
yolk sac, the indicated number of HPP-CFC colonies derived from 3 yolk
sacs were individually replated into HPP-CFC culture and secondary
colonies were scored after 7 days of incubation.
Flow cytometry
Cells in HPP-CFC culture were resuspended at
1 × 106/100 µL phosphate-buffered saline (PBS)
containing 1% fetal bovine serum (FBS), stained for 30 minutes at
4°C with the phycoerythrin-conjugated CD11b mouse antibodies
(Pharmingen, San Diego, CA), and analyzed.
Semiquantitative RT-PCR
Total RNA was isolated from EBs or HPP-CFCs using Trizol (Gibco
BRL, Gaithersburg, MD) according to the manufacturer's
instructions and then treated with DNase (Promega, Madison,
WI). Reverse transcription-polymerase chain reaction (RT-PCR)
was performed by using the mRNA selective PCR kit (Takara Shuzo,
Japan). A 10-fold dilution of each sample was PCR amplified to
achieve signals within the linear amplification range. Cycles for each
primer pair were empirically determined so as to yield product within
the early exponential phase of synthesis to ensure comparative
analyses. The gene primers, selected to cross introns where possible,
are listed in Table 1. All the genes were
analyzed on more than one occasion using cDNA from independently
derived RNA samples.
Statistical analyses
Values shown are the mean ± SD. Significant differences
between groups were evaluated using the Student t test and
P < .05.
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Results |
Increased frequency and replating potential of HPP-CFCs within E9.0
to E9.5 Smad5/ yolk sac
It has been previously reported that when assayed in 7-day CFC
cultures containing KL, IL-3, IL-6, and Epo, E9.5
Smad5/ yolk sac can give rise to an elevated number of
CFU-GMs compared with wild-type littermates.13 To further
evaluate the influence of Smad5 disruption on more primitive
progenitors in vitro, HPP-CFCs within the yolk sac were identified with
a distinct cytokine cocktail composed of KL, IL-3, IL-11, GM-CSF, and
Epo proven to favor the macroscopic colony growth. Single-cell
suspensions made from E9.0 to E9.5 yolk sac were plated into the
HPP-CFC culture and macroscopic colonies were scored according to
described criteria after 14 days of incubation. As shown in Figure
1A, the total number of HPP-CFCs from
Smad5/ yolk sac was twice that from wild-type and
heterozygous embryos. Thereafter, single-cell suspensions made from
individual HPP-CFC colonies plucked after 14 days of primary culture
were replated into secondary HPP-CFC cultures to compare the replating
potential among the 3 genotypes. After 7 days of secondary culture, a
similarly elevated number of colonies was found in the
Smad5/ group (Figure 1B). Cells from these secondary
colonies were examined by May-Giemsa staining and showed no morphologic
abnormalities. These results indicate that the Smad5 gene
may be involved in negative regulation of frequency and regeneration
capacity of HPP-CFCs during early embryonic hematopoiesis.
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| Figure 1.
Increased frequency and replating potential of HPP-CFCs
from Smad5/ yolk sacs at E9.0 to 9.5.
Dispersed yolk sac cells were cultured in HPP-CFC medium and the number
of macroscopic colonies was counted after 14 days of culture (A). Then
individual plucked macroscopic colonies were resuspended and plated
into secondary replating system and the number of secondary colonies
was scored after 7 days of culture (B). Results of HPP-CFC assay were
from 5 wild-type, 8 Smad5+/, and 6 Smad5/
embryos and are expressed as means ± SEMs. Results of replating
assay were obtained from 36 wild-type, 36 heterozygous, and 72 Smad5/ HPP-CFC macroscopic colonies of 3 yolk sac
cultures, respectively, and are expressed as means ± SEMs.
*represents data found to be significantly different from the
corresponding control values (P < .05).
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Elevated incidence of hematopoietic EBs from
Smad5/ ES cells
Dissection of the direct effect of Smad5 disruption on
hematopoietic development in vivo is complicated by its influence
on nonhematopoietic tissues. In E9.5
Smad5/ yolk sac, mesenchyme apoptosis and defects
in angiogenesis have been reported, the latter contributing to the
observed perturbations of embryonic circulation and ultimately
interfering with the migration of hematopoietic progenitors between
extraembryonic yolk sac and embryo proper. To get more reliable
information in this study, we next used the in vitro ES cell
differentiation system validated as a well-defined model of early
embryonic hematopoiesis. Initially, the effect of Smad5 disruption on
hematopoietic EB formation was examined. On differentiation in
semisolid culture containing KL and IL-11, proven critical for
efficient hematopoietic commitment of EBs, ES cells of all the 3 genotypes could form hematopoietic EBs. After 7 days of culture, these
EBs contained primitive erythrocytes and by 12 days macrophages were
observed at the periphery of the EBs. Strikingly at this time,
Smad5/ hematopoietic EBs showed a densely aggregated
hematopoietic halo around the central cell mass, whereas only a few
erythrocytes and myeloid cells were scattered at the periphery of the
wild-type hematopoietic EBs (Figure
2A,B). More intriguingly, the incidences of hematopoietic EBs were 29%, 59%, and 87% within wild-type, heterozygous, and homozygous EB-forming cultures, respectively, therefore, demonstrating a gene-dosage effect (Figure 2C). These data
may implicate the Smad5 gene as an inhibitor on
hematopoietic EB generation.
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| Figure 2.
Increased incidence of hematopoietic EBs from
Smad5/ ES cells.
ES cells were plated into methylcellulose medium containing KL and
IL-11 to form EBs. Note the extending hematopoietic halo surrounding
the central cell mass of 85% Smad5/ EBs (A), whereas
approximately 29% wild-type EBs ruptured with a few scattered
hematopoietic cells (B) after 12 days of differentiation. Incidence of
hematopoietic EBs was scored and values shown above each column
represent the means ± SEMs from 3 independent experiments (C).
Significance was determined using the Student t test:
*represents data found to be significantly different from wild-type
control values (P < .05); #, data found to be
significantly different from heterozygous values
(P < .05). Original magnification for panels A and B,
× 40.
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Enhanced BL-CFC number along with SCL and flk-1 transcripts within
day 3.5 Smad5/ EBs
To further determine the effect of Smad5 loss on distinct
hematopoietic precursors, EBs differentiated for various periods of
time were analyzed. BL-CFCs, the in vitro counterpart of hemangioblast with hematopoietic and endothelial potential, were first investigated. Two previous independent observations of Smad5 null mutant embryos suggest the possible involvement of Smad5 gene in BL-CFC
development. One group has detected increased and ectopic expression of
flk-1, a VEGF receptor and necessary for expansion and migration of
hemangioblast in mutant embryos.14 In the other report,
simultaneous abnormalities of hematopoietic progenitors' pool and
angiogenesis are documented.13 Hence,
Smad5/ ES cells were assayed for their potential to
generate the VEGF-responsive blast colonies. Secondary plating of
dissociated day 3.5 EBs cells gave rise to 3 types of colonies:
secondary EBs, transitional colonies, and blast colonies as previously
reported by other groups.21,22 As shown in Figure
3A, these colonies could be readily
detected and distinguished according to established morphologic
criteria. In comparison to the previous studies we saw a reduced
efficiency of blast colony formation because we did not add the medium
conditioned by the EB-derived endothelial cell line, D4T, to our assay.
This medium has been shown to reproducibly increase the growth
potential of BL-CFCs, and this omission was therefore likely to be
responsible for the reduction of blast colonies we
observed.23 However, as shown in Figure 3B, we clearly
found that Smad5/ EBs generated approximately a 3-fold
higher number of blast colonies than wild-type EBs (38 ± 5.7 from
Smad5/ EBs versus 10 ± 1.9 from wild-type EBs).
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| Figure 3.
Increased BL-CFC number and altered expression of
hemangioblast-related genes within day 3.5 Smad5/ EBs.
(A) Day 3.5 EBs were dissociated and then plated in BL-CFC assay
culture containing VEGF and KL. After 5 days of growth, 3 types of
colonies, secondary EBs, transitional colonies, and blast colonies were
observed. These colonies could be readily detected and distinguished
according to established morphology criteria. Original magnification
for panel A, × 100. (B) The number of blast colonies shown represents
the means ± SEMs from 3 independent experiments. *represents data
found to be significantly different from the wild-type control values
(P < .05). (C) Expression of Smad5 and
hemangioblast-related genes were defined by RT-PCR analysis of day 3.5 EBs. The arrows on the top of the photo in panel C represent the
10-fold dilution of samples.
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To further define the molecular change within Smad5/
EBs, the gene expression patterns of day 3.5 EBs were investigated
(Figure 3C). In agreement with the ubiquitous expression pattern of
Smad5 gene from gastrulation onward in embryos, its
transcripts were detected in day 3.5 wild-type EBs. Brachyury, coding
for a transcription factor expressed in mesoderm cells along the
primitive streak,24 was detected at a lower level in
Smad5/ EBs. In contrast, flk-1 was detected at a higher
level in mutant EBs, consistent with that in the Smad5/
yolk sac. Furthermore, SCL, a helix-loop-helix transcription factor expressed in both endothelial and hematopoietic lineages and
essential for hemangioblast and hematopoietic
specification,22,25,26 was also up-regulated. It is
suggested from previous findings that mesoderm cells fated to
hematopoietic and endothelial lineages may down-regulate Brachyury and
up-regulate flk-1 and SCL as they migrate to extraembryonic
sites.27 The collective alterations of Brachyury, flk-1,
and SCL that we have observed here in the Smad5/ EBs
may suggest an accelerated switch from mesoderm toward hematopoietic lineage and possibly contribute to the enhanced frequency of BL-CFCs within Smad5/ EBs. Thus, these findings suggest that
the negative role of Smad5 gene in regulating BL-CFC
development may be important at the point of mesodermal cell specification.
Smad5 gene was required for inhibition of embryonic
globin expression by TGF-1
As shown in Figure 4A, precocious
expression of embryonic H1 globin was detected within
Smad5/ day 3.5 EBs. Furthermore, the mutant EBs also
displayed enhanced expression of both GATA-1, indispensably required
for yolk sac primitive erythropoiesis, and Epo receptor.28
It has been reported that strengthened TGF- signaling by either
enforced expression or additional TGF-1 can significantly delay and
decrease the embryonic globin expression in cystic EBs formed in liquid
cultures.29 To determine if the precocious globin
expression within mutant EBs was due to the loss of the inhibitory
effect by TGF-1, we added TGF-1 to the EB-forming cultures and
compared such inhibition at day 4 of differentiation, the exact time
point for initial appearance of embryonic globin within wild-type EBs.
As shown in Figure 4B, TGF-1 was found to greatly decrease the
expression of H1-globin and  -globin within
wild-type EBs, whereas Smad5/ cells were not affected,
demonstrating that Smad5 gene was required for the negative
regulation by TGF-1 on embryonic globin expression at the
transcription level during primitive erythropoiesis.
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| Figure 4.
Investigation of embryonic globin gene expression by
RT-PCR within day 3.5 and 4.0 EBs.
The arrows on the top of each photo represent the 10-fold dilution of
samples. Hematopoietic-related genes were defined in collected day 3.5 EBs (A) and day 4 EBs (B). +/+T and  /T in panel B denote samples in
which additional TGF-1 (2 ng/mL) was supplemented to
wild-type and mutant EB-forming cultures.
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Loss of Smad5 gene resulted in defective
proliferation of Ery/Ps
The first detectable unilineage hematopoietic precursors within
EBs are Epo-responsive Ery/Ps, a population comparable to the blood
cells detected in mouse E7.5 yolk sac. To investigate the effect of
Smad5 disruption on this population, day 4, 5, and 6 EBs were
dissociated into single cells and cultured in methylcellulose medium
containing Epo to grow Ery/P colonies. At day 4, Ery/P colonies were
detectable only within wild-type EBs. At day 5 and day 6, such colonies
appeared within Smad5/ EBs, but the number was
significantly lower than that of wild type (Figure
5A). These results demonstrate that
Smad5 gene may be required for proliferation of Ery/Ps.
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| Figure 5.
Identification of Ery/Ps within wild-type and
Smad5/ EBs.
(A) EBs were dissociated at day 4 to 6 and replated into
cultures containing Epo favoring the growth of primitive erythroid
colonies. (B) The inhibitory effect of TGF-1 added to Ery/P colony
assays at final concentrations of 0.3 and 2 ng/mL. Numbers of Ery/P
colonies were scored after 5 to 7 days of culture. Results shown
(means ± SEMs) are from 3 independent experiments. Significance
was determined using the Student t test. *represents
data found to be significantly different from wild-type control values
(P < .05).
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To determine the effect of TGF-1 on the colony formation by Ery/Ps,
TGF-1 was directly added to Ery/P cultures containing Epo. As shown
in Figure 5B, wild-type and Smad5/ Ery/Ps displayed a
similar response to the inhibition of TGF-1. Thus, Smad5
gene was probably not involved in mediating the inhibitory signal of
TGF-1 on Ery/P colony generation.
Disruption of Smad5 gene led to increased frequency
of HPP-CFC within day 6 EBs
Next, HPP-CFCs within day 6 EBs were examined. As illustrated in
Figure 6C for the results averaged from 3 representative experiments, the frequency of HPP-CFCs and CFU-mix was
profoundly elevated within day 6 Smad5/ EBs compared
with that of wild-type EBs. A gene-dosage effect was also noted on
these 2 progenitor populations, with Smad5+/ EBs (10.0 HPP-CFCs and 17.9 CFU-mix) producing more colonies than wild type (4.2 HPP-CFCs and 8.9 CFU-mix) but fewer than Smad5/ EBs
(20.7 HPP-CFC and 50.0 CFU-mix). However, the frequency of CFU-GMs
seemed unaffected. Another interesting observation was the precocious
emergence of megacaryocyte CFUs within Smad5/ day 6 EBs
when cultured in medium supplemented with only thrombopoietin (Tpo). However, terminal differentiation of cells within the HPP-CFC cultures appeared unaffected. Specific lineages such as erythroid, granulocyte, and macrophage lineage of Smad5/ origin
were similar in morphology to those of wild-type origin examined by
May-Giemsa staining. To confirm the visual impression, the cellular
content of HPP-CFC culture was analyzed by flow cytometry. Similar
percentage of CD11b+ myeloid cells was detected in
wild-type and mutant cultures. Together, these results suggest that
disruption of Smad5 gene resulted in greatly enhanced
proliferation of definitive hematopoietic progenitors at an early stage
but the subsequent differentiation and maturation were not blocked.
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| Figure 6.
Identifications of HPP-CFC and primitive
progenitor-related gene expression within day 6 wild-type and
Smad5/ EBs.
HPP-CFCs derived from day 6 EBs of wild-type (A) and
Smad5/ (B) ES cells were able to generate macroscopic
colonies in vitro. Macroscopic colonies by HPP-CFCs are shown by white
arrows; secondary EBs are shown by white arrowheads. Grid lines on the
dishes are spaced 2 mm apart. Note the solid and round morphology of
secondary EBs, whereas the HPP-CFCs displayed a looser morphology and
often with red hemoglobin. The actual number of HPP-CFC,
erythroid mix (CFU-mix), and CFU-GM colonies derived from day 6 EBs are
shown in panel C. The results are shown as means ± SEM from 3 representative experiments; the other 2 experiments demonstrated
similar results. Significance was determined using the Student
t test. *represents data found to be significantly different from
wild-type control values (P < .05); #, data found to be
significantly different from Smad5+/ values
(P < .05). The patterns of gene expression were defined
using day 6 EBs by RT-PCR (D). The arrows on the top of the photo in
panel D represent the 10-fold dilution of samples.
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Thereafter, the gene expression patterns of day 6 EBs were examined
with particular regard to stem cell-related transcription factors.
GATA-2 is a zinc-finger transcription factor with probable functions in
driving early progenitor expansion,30,31 whereas AML1,
encoding the DNA-binding  subunit of core binding factor (CBF, also
known as PEBP2), is required for generation of all definitive
hematopoietic lineages.32,33 As shown in Figure 6D, day 6 Smad5/ EBs displayed increased expression of SCL and
AML1, which were likely responsible for increased numbers of HPP-CFCs
within mutant EBs. Nevertheless, expression of GATA-2 was greatly
decreased. The implication of this contradictory alteration of these
stem cell-related transcription factors within mutant EBs remains to be determined.
Reduced sensitivity of Smad5/ HPP-CFCs to
inhibition by TGF-1
The increased number of macroscopic colonies generated by early
progenitors within day 6 EBs largely resembled previous findings using
TGF-1 antisense oligonucleotides or anti-TGF-1 serum in human
bone marrow and cord blood.34,35 Therefore, the response of wild-type and Smad5/ HPP-CFCs to TGF-1 was
tested (Figure 7). At concentrations of between 0.03 and 0.3 ng/mL TGF-1, macroscopic colony formation was
inhibited within wild-type EBs in a concentration-dependent manner and
this inhibition was partly but significantly abrogated by the loss of
Smad5. However, whereas the Smad5/ HPP-CFCs were
significantly resistant to TGF-1-induced inhibition, higher doses
(up to 2 ng/mL TGF-1) could completely inhibit the formation of
macroscopic colonies in Smad5/ cultures. These results
suggest that in addition to Smad5, there are other signaling cascades
by which TGF-1 may exert the inhibitory effect on HPP-CFCs. Similar
but less efficient inhibition was also observed when the same dosage of
TGF-2 was added (data not shown).
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| Figure 7.
Reduced sensitivity of Smad5/ HPP-CFCs
to TGF-1 inhibition.
The inhibitory effect of TGF-1, added to HPP-CFC assay at final
concentrations of 0.03, 0.1, 0.3, and 2 ng/mL, is shown. These results
are from a representative experiment; 4 other independent experiments
demonstrated similar results.
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Profoundly elevated replating potential of day 6 Smad5/ EB-derived HPP-CFCs
Qualitatively, HPP-CFCs are capable of forming secondary colonies.
To identify such functional features of ES cell-derived HPP-CFCs, we
carried out preliminary investigations on replating potential of day 6 wild-type EB-derived HPP-CFCs. All the HPP-CFCs we individually plucked
were capable of forming secondary CFU-GM colonies composed mainly of
neutrophils, mast cells, and macrophages. About 60% of HPP-CFC
colonies could form secondary HPP-CFC colonies, whereas approximately
35% of HPP-CFC colonies could produce secondary definitive erythroid
colonies (BFU-Es and CFU-Es). The ratio of secondary CFU-mix
colonies was relatively lower, nearly 30%. The cellular content of 24 secondary HPP-CFC colonies was examined by May-Giemsa staining and all
the colonies uniformly contained neutrophils, mast cells, and
macrophages resembling in constitution those from E9.5 yolk
sac-derived HPP-CFCs described previously.16 These
results showed that ES cell-derived HPP-CFCs were multipotential.
Next, to investigate the effect of Smad5 disruption on the regeneration
capacity of HPP-CFCs, we plated cells from day 6 EB-derived individual
or bulk HPP-CFCs into secondary HPP-CFC culture and secondary HPP-CFCs
were scored after a further 14 days of incubation. As shown in Table
2, each primary wild-type HPP-CFC could
give rise to 3.4 secondary HPP-CFCs on average. In contrast, 19.7 secondary HPP-CFCs were generated from each primary
Smad5/ HPP-CFC, approximately 4- fold higher than that
of wild type. Thereafter, the secondary HPP-CFCs were individually
picked and mixed and 1 × 105 cells were plated for
tertiary HPP-CFC assay. Although no tertiary HPP-CFC colonies were
detected in wild-type culture, a significant number of tertiary
macroscopic colonies were observed within the Smad5/
culture.
Another discrepancy was detected in an aspect of constitution of
secondary HPP-CFC colonies. Most secondary Smad5/
HPP-CFCs were of multilineage potential consisting of erythrocytes, granulocytes, mast cells, and macrophages (Figure
8B,F,J). In contrast, erythrocytes were
absent from about 80% of secondary wild-type HPP-CFC colonies (Figure
8A,E,I). The tertiary mutant HPP-CFC colonies were also composed of
multilineage cells (Figure 8C,D,G,H,K,L). Together, these results
indicate that Smad5/ HPP-CFCs from day 6 EBs harbored
extensive self-renewal capacity, whereas this capacity of wild-type
HPP-CFC was relatively limited.
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| Figure 8.
Secondary and tertiary colonies derived from primary wild-type and
Smad5/ HPP-CFCs in replating assay.
Most of the Smad5/ HPP-CFC colony was able to form
secondary HPP-mix consisting of erythrocytes, granulocytes, mast cells,
and macrophages (B,F,J), whereas wild type generated almost all
HPP-GMs (A,E,I). In tertiary cultures, no macroscopic HPP-CFC colony
was detected in wild-type culture (C,G,K), whereas the tertiary
macroscopic HPP-CFC colonies, most of which were of mixed cellular
content, were readily observed in mutant culture (D,H,L). Original
magnifications: panels A-D, × 40; panels E-H, × 200; and panels
I-L, × 1000.
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To define genetic correlation with the increased regeneration capacity
of mutant HPP-CFCs, we performed RT-PCR on primary HPP-CFCs. Compared
with EBs, the gene expression profile of HPP-CFCs was far more specific
to define the status of primitive multipotential progenitors. As shown
in Figure 9, wild-type and mutant
HPP-CFCs expressed elements of TGF- signaling cascade including
TGF-1, TRI, and TRII, thereby suggesting a possible
regulation loop of hematopoietic progenitors by autocrine TGF-1.
Smad5/ HPP-CFCs contained profoundly enhanced
transcripts of GATA-2, AML1, GATA-1, EKLF, and comparable NF-E2. In
addition, IL-3 receptor and GM-CSF receptor were found at significantly
higher expression levels in Smad5/ HPP-CFCs, although
c-kit and Epo receptor were unaffected. Together, the implications of
these molecular findings were generally in agreement with the
increased regeneration capacity of Smad5/ HPP-CFCs.
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| Figure 9.
Altered gene expression within Smad5/
HPP-CFCs.
Macroscopic HPP-CFC colonies derived from day 6 wild-type and
Smad5/ EBs were individually plucked and mixed for
RT-PCR examination. The arrows on the top of each photo represent the
10-fold dilution of samples.
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Discussion |
In this study, we showed that disruption of Smad5 gene
led to increased frequency and regeneration capacity of HPP-CFCs within yolk sac and EBs, demonstrating that SMAD5 may negatively regulate the
proliferation and self-renewal of these early progenitors during
embryonic hematopoiesis. The involvement of Smad5 gene in
TGF- signaling pathway has been raised by previous genetic and
biochemical experiments. First, the resemblance of the Smad5 mutant
yolk sac phenotype to the TGF-1, TRI, and TRII knockouts is
fairly striking.36-38 Second, Smad5 has a unique
DNA-binding property that is similar to Smad3, which specifically
transduces signals for TGF- receptors.39 The most
direct investigation using antisense oligonucleotides has shown that
Smad5 is required for proliferative inhibition by TGF- on
hematopoietic progenitors from human bone marrow. Several lines of
evidence demonstrated here suggest that the escaping from TGF-1
modulation may contribute, at least in part, to the
aberrant behaviors of Smad5/ HPP-CFCs during
embryonic hematopoiesis. It has been reported that differentiating EBs
express a set of components involved in TGF-1 signaling pathway
including TGF-1, TRI, and TRII, suggesting that an autocrine
regulation exerted by TGF-1 may exist in embryonic
development.29,40,41 Here, we showed that these genes as
well as Smad5 gene were expressed in EB-derived HPP-CFC
colonies, thus, specifying such regulation loop to embryonic hematopoiesis. As reported, TGF-1 is preferentially either in vivo
or in vitro active on LTC-ICs and HPP-CFCs. Such a specific effect was
also observed on EB-derived HPP-CFC. However, sensitivity of
Smad5/ HPP-CFCs to inhibition by TGF-1 was
significantly reduced, partly in line with the finding using antisense
oligonucleotide to Smad5 gene in human bone marrow.
Nevertheless, it seems that Smad5 is not the sole signal transducer of
TGF-1 during embryonic hematopoiesis because a high concentration of
TGF-1 still can abrogate the proliferation of Smad5/
HPP-CFCs. In addition, an elevated number of HPP-CFC within
Smad5/ yolk sacs and EBs was similar to an alternative
approach using blocking antibodies or antisense oligonucleotides to
neutralize TGF-1 secreted by hematopoietic cells, and consequently
leading to increased numbers of macroscopic colonies in human bone
marrow and cord blood.34,35 Finally, the up-regulated
expression of IL-3 and GM-CSF receptors in Smad5/
HPP-CFCs fit well with the ability of TGF-1 to modulate the expression of a variety of cytokine receptors.42
Commonly, the inactivation of the TGF- signaling cascade in early
hematopoietic cells, which are normally quiescent, has been proposed to
result in their escaping from cell cycling inhibition and finally
malignant transformation. For instance, abnormalities in the expression
of TGF- receptors have been described in proliferative syndromes
including both early myeloid43,44 and lymphocytic leukemia.45,46 Likewise, either Smad mutations or Smad
functional inactivation have also been associated with malignant
transformation. Chimeric transcription factors, such as AML1/Evi-1 and
AML1/ETO resulting from t(3;21) and t(8;21) chromosomal translocations in chronic and acute myelogenous leukemia, have been documented to
block TGF- signaling by repression of Smad3
activity.47,48 Smad5 gene has been located at
human chromosome 5q31, which is commonly deleted in myelodysplastic
syndromes and acute myeloid leukemias (MDS/AML), thus is suspected as a
leukemia suppressor gene.49,50 Nevertheless, the aberrant
behavior of Smad5/ primitive progenitors was found
mainly in significantly increased self-renewal capacity with the
terminal differentiation unaffected, and, therefore differed to some
extent from that of AML1/Evi-1 and AML1/ETO, which ultimately cause
abnormal and blocked differentiation. More intriguingly, a gene-dosage
effect was observed in hematopoietic EB incidence and HPP-CFC assay,
which in some sense strengthened the specificity of Smad5
gene. This haploinsufficiency of Smad5 gene may be an
important clue for the speculation of Smad5 as a potent leukemia
repressor. Previous observation that no intragenic mutations are
detected in the remaining allele of leukemic patients and several human
leukemia cell lines prescreened for hemizygously loss of
Smad5 gene may suggest the possible correlation between such
haploinsufficiency and leukemogenesis. Hence, the hematopoietic characteristics of heterozygous Smad5+/ ES cells and
mouse deserve more comprehensive analysis.
Another interesting finding demonstrated that loss of Smad5
gene resulted in increased BL-CFC frequency concomitant with elevated transcriptional level of SCL and flk-1 gene, which are considered to be
specific markers of the hemangioblast.21,22 Which
signal mediated by SMAD5 is responsible for the negative regulation on BL-CFC development? The importance of BMP-4, the initially proposed upstream ligand of SMAD5, for hematopoietic development in the mouse
has been established from effects of its genetic deletion, which
disrupts mesoderm and blood cell formation in the yolk
sac.51 It is further verified in an ES cell
differentiation as well as Xenopus model, demonstrating the function of
BMP-4 in inducing or up-regulating the expression of specific
transcription factors including SCL, GATA-1, and GATA-2 together with
embryonic globins.52-54 Generally, BMP-4 may continuously
play pivotal and positive roles throughout the course of hematopoietic
commitment. Particularly, some previous data implicate its positive
involvement during BL-CFC commitment. In rhesus monkey ES cell
differentiation assay, BMP-4 is able to induce the expression of SCL,
indispensably required for hemangioblast commitment from mesoderm
cells, and to elevate the expression of flk-1 that is necessary for
expansion and migration of hemangioblast.53 Furthermore,
flk-1 is not detectable in EBs stably transfected with
dominant-negative BMP receptors.54 Unlike |
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Abstract
Introduction