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First published online 13 December 2006
doi: 10.1242/dev.02731
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1 Department of Pathology and Immunology, Washington University School of
Medicine, St Louis, MO 63110, USA.
2 Molecular Cell Biology Program, Washington University School of Medicine, St
Louis, MO 63110, USA.
3 Department of Pharmacology, University of Wisconsin Medical School, Madison,
WI, USA.
4 Center for Developmental Biology, University of Texas Southwestern Medical
Center, Dallas, TX, USA.
* Author for correspondence (e-mail: kchoi{at}wustl.edu)
Accepted 7 November 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Embryonic stem, Hemangioblast, BMP4, GATA2, Flk1 (Kdr1), Scl, Cell cycle
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Nishikawa et al.,
1998). In further determining molecular markers of the
hemangioblast, we recently showed that Flk-1+Scl+ (Scl:
Tal1 - Mouse Genome Informatics) cells derived from differentiating ES cells
are enriched for the hemangioblast (Faloon
et al., 2000; Chung et al.,
2002). In the developing embryo,
brachyury+Flk-1+ cells readily displayed hemangioblast
activity as defined by their in vitro potential to form Blast colonies
(Huber et al., 2004). High
hemangioblast activity was detected in the posterior primitive streak, whereas
distal primitive streak, the anterior region, the lateral domains, and the
yolk sac had little hemangioblast activity. These studies suggest that the
hemangioblast in the developing embryo arises within the primitive streak and
rapidly differentiates into its progeny, hematopoietic and endothelial
precursors. Thus, the bi-potentiality of the hemangioblast to generate blood
and endothelial cells appears to be rapidly lost once the hemangioblast is
formed, implicating a transient nature for the hemangioblast. Consistent with
this notion, the bipotentiality of the EB (embryoid body, in vitro
differentiated ES cells)-derived BL-CFC is highest when analyzed from the
earliest stages of BL-CFC formation (Choi
et al., 1998).
The mechanisms that regulate the development and differentiation of the
hemangioblast are poorly understood. BMP signaling is crucial for
hematopoietic and vascular development
(Larsson and Karlsson, 2005;
Miyazono et al., 2005;
Moser and Patterson, 2005).
Recently, we demonstrated that BMP4 was sufficient to generate
Flk-1+ mesoderm (Park et al.,
2004). Specifically, BMP4 was able to induce Flk-1+
cells from the brachyury+ stage of EB cells in serum free
conditions, suggesting that BMP4 specifies differentiation of
Flk-1+ mesoderm from brachyury+ mesoderm. The downstream
effectors that mediate BMP signaling are unknown. However, BMP4 signaling has
been implicated in activating expression of Gata2, an important
regulator of multipotent hematopoietic precursor cells. Utilizing
Xenopus animal cap assays, Maeno et al.
(Maeno et al., 1996) showed
that injection of bmp4 mRNA directly into animal caps activated
Gata2 gene transcription. Furthermore, Friedle and Knochel
(Friedle and Knochel, 2002)
showed that the transcriptional activation of Gata2 was translation
independent, as cycloheximide did not block Gata2 transcription. The
identification of a Bmp4 response element (BRE) in the 5' promoter
region of zebrafish gata2 that binds Smad1 and Oct-1 provides further
evidence for BMP4 mediated activation of gata2
(Oren et al., 2005).
In the current report, we undertook a global gene expression profiling
approach to identify BMP4 targets during hematopoietic and vascular
development. We compared gene expression profiles between
Flk-1+Scl+ cells, a subset of Flk-1+ mesoderm
that contains the putative hemangioblast cell population, and their
differentiated progeny, Blast cells (Choi
et al., 1998). Based on bioinformatic analysis of patterns of gene
expression, the BL-CFCs resembled other multipotential stem cell populations.
At the individual gene level, we noted that Gata2 was enriched in
BL-CFCs. Our present studies indicate that Gata2 is rapidly induced
by BMP4 and that enforced GATA2 upregulates Bmp4 expression. Enforced
Gata2 expression also induced Flk-1+ and Scl+
cells in the absence of any added factors. GATA2 occupied regulatory elements
at the Bmp4 and Scl loci in differentiating EBs.
Importantly, the increase in Flk-1+ and Scl+ cell
generation resulted in an expansion of Blast as well as primitive erythroid
and endothelial cell progenitors. Systematic modulation of Gata2
provided strong evidence for the transient nature of the hemangioblast.
Additionally, enforced Gata2 expression in the primitive erythroid
progenitor revealed its essential role in promoting cell cycle progression.
Collectively, we provide compelling evidence for GATA2 function in
hemangioblast generation and differentiation.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Briefly, cells were differentiated in IMDM containing
15% differentiation-screened FCS, 1% L-glutamine, 50 µg/ml
ascorbic acid and 4.5x10-4 M MTG. Serum-free conditions
substituted KNOCKOUT SR (Gibco BRL) for FCS and contained 5% PFHM II (Gibco
BRL). GATA2 was induced in iGATA2 cells with 0.3 µg/ml doxycycline (Dox).
BMP4 (R&D Systems) was added at 5 ng/ml. Noggin (R&D Systems) was used
at 10 ng/ml. Blast colonies were generated by harvesting cells at the
indicated day, dissociating in trypsin, passaging through a 20G needle four to
six times and plating in the presence of VEGF (5 ng/ml), kit ligand (1%
conditioned media) and D4T endothelial cell conditioned media (25%) and with
or without Dox for iGATA2 cells. Erythroid cells were generated in
methylcellulose in the presence of 10% plasma-derived serum (PDS, Antech, TX),
5% PFHM II, ascorbic acid (12.5 µg/ml), L-glutamine (2 mM),
transferrin (300 µg/ml, Boehringer Mannheim) and 2 units/ml EPO (Amgen,
Thousand Oaks, CA)
GeneChip analysis
GeneChip results were analyzed in dChip
(Li and Wong, 2001;
Zhong et al., 2003). Median
chip intensity and percent present call were similar across all chips and were
within normal ranges; gene expression differences were determined using a 90%
confidence interval of >1.2-fold and above, and a baseline to experimental
intensity difference of >50. Genes enriched in each cell population (Blast
or BL-CFC) were functionally categorized using GOurmet to determine
distribution of the Gene Ontology (GO)
(http://www.geneontology.org/)
terms associated with each gene in the list
(Doherty et al., 2006). For
Fig. 1A, all genes in either
the BL-CFC- or Blastenriched lists that fell under the GO term `regulation of
transcription, DNA-dependent' were determined and a heat map of these genes
was prepared using the Hierarchical Clustering feature of dChip. All GeneChip
data will be uploaded to Gene Expression Omnibus (GEO) for archiving and
sharing purposes.
GOurmet (Doherty et al.,
2006) was also used to determine the distribution of all GO terms
in a series of previously published expression profiles of stem cell
populations with their paired differentiated progeny. In all cases, the lists
of stem cell-enriched genes were determined by acquiring the original .DAT or
.CEL chip files and simultaneously comparing replicate progenitor chips to
replicate progeny chips using the same threshold criteria as for BL-CFC and
Blast. GO distributions were determined for each cell population, and the
lists were thereby hierarchically clustered
(Fig. 1C). For further details
of this approach see Mills et al. (Mills
et al., 2002) and Doherty et al.
(Doherty et al., 2006).
Gene expression analysis
RNA was purified using TRIzol (Invitrogen, CA), following the
manufacturer's protocol. Total RNA (1 µg) was treated with DNase I and then
annealed to 50 ng of random primers at 65°C. Reverse transcription was
carried out with RNase H- Superscript II reverse transcriptase, 10
mM DTT, RNaseOUT (all from Invitrogen), and 1 mM dNTPs at 42°C for 1 hour.
Reactions were diluted with DEPC-TE to 95 µl followed by heat inactivation
at 65°C for 15 minutes. Quantitative PCR (qPCR) reactions (15 µl)
contained 1 µl cDNA, 7.5 µl of iQ SYBR Green Supermix (BioRad), 0.15
µl uracil DNA glycosylase (Invitrogen) and 200 nM each forward and reverse
primers. Primer sequences are given in
Table 1. The effect of BMP4
addition on Gata2 expression was tested by growing R1 ES cells for 2
days in serum-free media (formulation as above). EBs were harvested on day 2.
Cells were collected, washed once with 10 ml IMDM, resuspended in 1 ml IMDM,
and incubated on ice for 20 minutes. Recombinant BMP4 (50 ng; R&D Systems)
was added along with either 10 µg/ml cycloheximide or DMSO as a vehicle
control and cells were incubated for 5 minutes on ice before being transferred
to 37°C for the appropriate time period. After qRT-PCR, the expression
level of Gata2 in untreated EBs was normalized as 1, after being
normalized against Gapdh values.
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).
Day 4, 5 or 6 A2Lox EBs were harvested and crosslinked with 1% formaldehyde at
room temperature for 10 minutes. Cells were lysed and chromatin slurry was
incubated with Rabbit IgGs (PI) (Santa Cruz), GATA1, or GATA2 antibodies
(Im et al., 2005). Genomic
material was recovered for assessment by qPCR using the primers listed in
Table 1.
Flow cytometry
For all FACS analyses, EBs were dissociated in 7.5 mM EDTA in PBS for 1.5
minutes at 37°C. Cells were then dissociated by passaging through a 20G
needle 5-7 times prior to being resuspended in washing/staining buffer (4% FCS
in PBS) and counted. Cells were resuspended at 5x106 cells/ml
and plated into individual v-shaped wells of a 96-well plate at
5x105 cells per well. Cells were incubated with primary
antibodies for 15 minutes on ice, washed three times and then incubated on ice
in the dark with secondary antibodies for 15 minutes. Cells were washed three
times and analyzed using a Becton Dickinson FACS Caliber. For cell-sorting
experiments, a MoFlo high speed flow cytometer (Dako, Fort Collins, CO) was
used. The primary antibody was a biotinylated antihuman CD4 (Caltag, 1:500).
Secondary antibodies include streptavidin-allophycocyanin (SP-APC, Pharmingen,
1:1000), anti-Flk-1-phycoerythrin (Flk-1-PE, Pharmingen, 1:200), anti-CD31-PE
(CD31-PE, Pharmingen, 1:200), anti-VE-Cadherin (Pharmingen, 1:200) and
anti-Tie2-PE (Tie2-PE, Pharmingen, 1:200).
Biochemical analysis of iGATA2 cells
To examine the induction of GATA2 protein in iGATA2 cells, cells were
differentiated in serum and the indicated amount of Dox was added at D2 of EB
formation and cells were collected at D4 of EB formation. Subsequent steps
were performed as previously described
(Park et al., 2004). The GATA2
antibody used was the same as in the above ChIP experiments.
BrdU uptake assays
Assays were performed with the Cell Proliferation Biotrak ELISA System 2
(Amersham Biosciences). On day 2 or 3 of EryP formation, cells were recovered
by cellulase treatment. Cells (3x103 and
1x104) were labeled with BrdU and cultured overnight in media
containing IMDM, 15% PDS and 2 units/ml EPO with or without Dox. Cells were
prepared according to the manufacturer's protocol for examination on a plate
reader. A `no BrdU' condition was set up to determine the background
absorbance of the cells and subsequently subtracted from all values. `No Dox'
absorbance values were normalized to 1 and the absorbance of the Dox-treated
cells was normalized against the control value to give a relative BrdU
incorporation level.
EB sprouting assays
Assays were based upon aortic ring assays
(Nicosia and Ottinetti, 1990a;
Nicosia and Ottinetti, 1990b;
Wang et al., 2004). Growth
Factor Reduced Matrigel (BD Biosciences) was diluted 1:1 with IMDM containing
2% FCS and 50 µl was plated into wells of a 96-well plate and allowed to
polymerize for 40 minutes at 37°C. A single D6 EB was recovered from SR
differentiation media and placed on the Matrigel bed, and an additional 50
µl was then plated on top of the EB. The upper layer was incubated for 40
minutes at 37°C before media containing IMDM, 2%FCS and 30 ng/ml VEGF was
added. EBs were cultured for an additional 6 days and the sprouting pattern
was scored. For LDL uptake studies, the upper matrigel layer was omitted and
DiL-Ac-LDL (Biomedical Technologies, Inc.) was added at 10 µg/ml and
incubated for 3 hours. Medium was removed and EBs washed three times with
fresh media and then images captured.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). This allowed us to define patterns across stem and
differentiated cell datasets originating from several different investigators,
each using different methods. Of the hemangioblast-enriched genes, about 30%
were classified under the GO term `nucleus', 15% each represented
`DNA-binding' and `regulation of transcription', and 5-10% each were `nucleic
acid-binding' and `RNA-binding' genes (Fig.
1B). The enrichment in those GO terms caused the hemangioblast
cell population to cluster with profiles of other stem cell populations
including hematopoietic, neural and gastric stem cells
(Fig. 1C). Thus, the analysis
suggested considerable similarity in the types of genes expressed by stem
cells of multiple tissues and, moreover, revealed the previously undescribed
stem cell phenotype of the BL-CFCs.
Gata2 in differentiating ES cells
Our array data also provided us with potential targets that may be
important for regulation of hemangioblast specification from mesoderm. We
observed high expression of Gata2 in the hemangioblast relative to
its more committed progeny (Fig.
1A). Our previous data showed that BMP4 signals were able to
induce Flk-1+ as well as Scl+ cells from ES cells in
culture (Park et al., 2004).
Thus, we tested if Gata2 was a direct target of BMP4. To this end, we
differentiated ES cells in serum-free [i.e. serum-replacement (SR)] media for
2 days, treated them with BMP4 and cycloheximide and then harvested EBs after
an hour of stimulation to examine Gata2 expression. As shown in
Fig. 2A, BMP4 treatment led to
a large increase in Gata2 mRNA within 1 hour, and this increase was
maintained in the presence of cycloheximide. This demonstrated that
Gata2 was a direct target of BMP4 signaling. Importantly,
Flk1 and Scl were not upregulated following an hour of BMP4
treatment (not shown).
Next, we examined expression kinetics of Gata2 in differentiating
ES cells, in relationship to other key differentiation markers including
Rex1 (Zfp42 - Mouse Genome Informatics), brachyury,
Flk1 and Scl. To this end, A2Lox ES cells, a derivative of
E14Tg2a ES cells (see below), were differentiated for 5 days in
serumcontaining differentiation media and harvested at each day for qRT-PCR
analysis. The ES gene Rex1 has been shown to be rapidly downregulated
as ES cells begin to differentiate (Faloon
et al., 2000). As expected, Rex1 expression was rapidly
downregulated after day 0 (D0) and expression was lost after D2
(Fig. 2B). Concurrently at D2
of differentiation, brachyury was detected and then peaked at D3. Shortly
after the generation of brachyury+ mesoderm, Gata2 and
Flk1 were induced and their expression rapidly increased, peaking at
D4. Lastly, Scl expression was detectable shortly after Flk1
and peaked at D5 (Fig. 2B).
Gata1 and cKit began to be expressed at D4 and their
expression increased at D5 and thereafter (not shown). These data indicate
that as A2Lox ES cells differentiate and generate mesoderm, Gata2 and
Flk1 are amongst the first genes expressed
(Fig. 2B). Thus, consistent
with previous reports from Xenopus models, Gata2 is rapidly
induced by BMP4 from brachyury+ mesoderm and precedes the
generation of more committed hematopoietic genes in the ES/EB system.
The specification and differentiation of Flk-1+ mesoderm by GATA2
To determine if Gata2 plays a role in hemangioblast development
and differentiation, we utilized the ES/EB system. Given the expression
kinetics of Gata2 in differentiating EBs, we sought to induce GATA2
at specific stages of EB generation and differentiation to accurately address
the role that GATA2 plays once ES cells have differentiated to mesoderm
(brachyury+). To this end, we targeted the tet-responsive locus of
A2Lox cells [an E14Tg2a-based ES cell line, similar to Ainv15
(Kyba et al., 2002), but with
superior targeting efficiency (M.K., unpublished)] with the Gata2
cDNA. To facilitate detection of Scl-expressing cells, we also
knocked-in a non-functional human CD4 (hCD4) into the Scl locus, as
described previously (Chung et al.,
2002) (Fig. 2C).
Once the correct targeting event was confirmed by a tet-responsive
locus/cDNA-vector-specific PCR, inducible Gata2 expression was
verified by adding doxycycline (Dox) to differentiating EB cells. As shown in
Fig. 2D, exogenous GATA2
protein was readily detectable in day 4 EBs with Dox concentrations as low as
0.03 µg/ml. For all subsequent studies with this inducible GATA2 (iGATA2)
ES cell line, we applied 0.3 µg/ml of Dox, at which concentration no
cytotoxicity was observed (not shown).
Employing these cells, we first studied the GATA2-mediated transcriptional
program. Cells were differentiated in SR media, GATA2 was induced at D2 of
culture (brachyury+ stage), differentiated for an additional 2 days
and the gene expression profile was examined. To assess the genetic program
mediated by GATA2, we utilized a candidate gene approach to examine a number
of specific cell types including mesoderm, hematopoietic, cardiogenic,
endoderm and ectoderm (Fig.
3A). The expression of brachyury was downregulated in
GATA2-induced cells as compared with non-induced control cells. As ES cells
differentiated in SR media have a longer window of brachyury expression in the
absence of differentiating cues such as BMP4
(Park et al., 2004), we
reasoned that the expression of GATA2 accelerated the differentiation of
brachyury+ mesoderm into more-differentiated mesodermal
derivatives. Accordingly, the expression of Bmp4 and
Pdgfr
was increased, arguing that GATA2 promoted the
differentiation of mesoderm into its derivatives
(Fig. 3A). We examined a set of
genes that would provide insight into how GATA2 regulates the differentiation
of Flk-1+ mesoderm. Specifically, Flk1, Scl, Gata1 and
Kit, were all expressed at higher levels when GATA2 was induced
(Fig. 3A). Additionally, the
cardiogenic genes Hand2, Nkx2-5 and Gata4 were also
upregulated upon GATA2 induction, consistent with Flk-1+ mesoderm
giving rise to cardiac tissues (Iida et
al., 2005; Kouskoff et al.,
2005; Wang et al.,
2006). Lastly, induction of GATA2 led to a marked reduction in the
expression of the endodermal genes Hnf3ß (Foxa2-Mouse
Genome Informatics), Hnf4 and Ttr as well as the ectodermal
genes Fgf5 and Neurod1. In the context of mesoderm
differentiation, GATA2 has a positive effect upon hematopoietic and
cardiogenic mesoderm formation and suppresses endoderm and ectodermal
lineages.
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)
(http://genome.lbl.gov/vista/index.shtml)
as well as the rVista program for identification of transcription
factor-binding sites (Loots et al.,
2002). We identified three conserved GATA-factor-binding sites
(WGATAR, where W is A or T, and R is A or G)
(Bresnick et al., 2005) between
mouse and human: the first, 1034 bp upstream of the first exon (-1034 site);
the second, 392 bp upstream of the first exon (-392 site); and an inverted
repeat pair (Tandem GATA Enhancer, or TGE, site) located 3.9 kb into the first
intron (Fig. 3B). This inverted
repeat is similar to the pair seen in the Gata1 locus capable of
conferring Gata1 activity in eosinophils
(Yu et al., 2002). Moreover,
this TGE site shows tremendous conservation amongst Mouse, Human, Rat, Dog,
Monkey and Chimp (Fig. 3B). To
examine whether these conserved sites were relevent in vivo, we used
quantitative ChIP to measure GATA2-chromatin interactions in the parental
A2Lox ES cell line. Specifically, A2Lox ES cells were differentiated for 4
days in serum and crosslinked to capture protein-DNA binding interactions.
After immunoprecipitation with an anti-GATA2 antibody, the recovered genomic
material was subjected to quantitative PCR. As shown in
Fig. 3C, GATA2 occupied the TGE
during stages of A2Lox ES cell differentiation. To confirm the relationship
between GATA2 and Bmp4 expression, we took advantage of the knock-in
SclhCD4/+ allele in iGATA2 ES cells and reasoned that
hCD4+ cells generated by Dox addition (see below) could be used to
isolate cells that expressed exogenous GATA2. As shown in
Fig. 3D, in cells sorted as
hCD4+, there was a large enrichment of both Gata2 and
Bmp4, indicating that the expression levels of Bmp4
correlated to Gata2 expression. This suggests a unique regulatory
mechanism whereby BMP4 is capable of inducing Gata2 expression and
GATA2 in turn contributes to the direct activation of Bmp4.
|
), the addition of BMP4 was sufficient to generate
Flk-1+ cells. More importantly, GATA2 alone could also generate
Flk-1+ cells. When GATA2 was induced in the presence of BMP4, the
Flk-1+ cell generation was similar. Furthermore, GATA2 could still
generate Flk-1+ cells when BMP4 was blocked by noggin. We compared
the human KDR and mouse Flk1 loci for regions of homology
and discovered three conserved GATA-binding sites within 10 kb upstream of the
Flk1 gene. However, ChIP assays did not indicate in vivo occupancy by
either GATA2 or GATA1. Collectively, these data argue that GATA2 is a
downstream component of BMP4-mediated signals that regulate Flk-1+
mesoderm generation, but that such regulation is not mediated by the 10 kb
proximal region of Flk1.
Hemangioblast, hematopoietic and endothelial cell generation by GATA2
To determine the role of GATA2 in the generation of Scl+ cells
and their progeny, iGATA2 cells were differentiated in serum, SR, or SR
containing BMP4 in the presence or absence of Dox (at D2 of culture), and
hCD4+ cells were assessed by FACS analyses on D4 or D5. Previous
work has shown that BMP4 could generate a moderate level of hCD4+
cells in the absence of serum but that a more robust level of hCD4+
cells could be generated when BMP4 and vascular endothelial growth factor
(VEGF) were added together (Park et al.,
2004). Fig. 4A
shows that the induction of GATA2 in the absence of serum resulted in a high
level (
50%) of hCD4+ cells at D5, whereas less than 3% of
cells became hCD4+ in SR media. Importantly, GATA2 induction did
not increase the expression of Vegf, arguing that GATA2 induction of
Scl+ cells is independent of VEGF signaling
(Fig. 3A). Consistent with
previous studies (Park et al.,
2004), the addition of BMP4 to the media was able to augment
generation of hCD4+ cells to 13%. There was no additive effect
between BMP4 and GATA2 on Scl expression, as the addition of BMP4 to cells
with enforced GATA2 expression did not change the generation of
hCD4+ cells as compared with that of GATA2 alone
(Fig. 4A,A').
|
) and
then determined which of these sites were conserved between human and mouse.
Strikingly, although there were abundant WGATAR sites in both species (27 in
human, 33 in mouse), only a single site was conserved between the two species
(Fig. 4B). This site,
identified in the human SCL gene as capable of binding GATA1 and
GATA2, is 37 bp upstream of the transcriptional start site of SCL
(Aplan et al., 1992). In the
mouse locus, this site resides 34 bp upstream of the transcriptional start
site. Importantly, the GATA site in the +19 enhancer region previously posited
to confer hematopoietic specificity during development
(Gottgens et al., 2002;
Gottgens et al., 2004) was not
a consensus WGATAR site in human. We examined the Scl locus during
differentiation in the parental A2Lox ES line, reasoning that
GATA-factor-binding may be developmentally dynamic in the control of
Scl. To this end, we performed quantitative ChIP analyses using D4,
D5 and D6 EBs and tested both GATA2 and GATA1 occupancy at the -34 promoter
site as well as the +19 enhancer site (Fig.
4C). At D4 of EB formation, which is the early stage of
Scl+ cell generation, GATA2 preferentially occupied the
Scl promoter. GATA1 was also detected at the promoter at D4 and
importantly, as differentiation progressed, GATA2 was displaced from the
promoter and GATA1 was bound at higher levels at D5 and D6. During all stages
of EB development, negligable signals were detected at the +19 enhancer
(Fig. 4C).
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;
Park et al., 2005). To
determine if GATA2 plays a role in the generation and differentiation of the
hemangioblast, we first differentiated J1(WT) and Gata2-/-
ES cells and replated these cells for Blast colonies. As shown in
Fig. 5A,
Gata2-/- cells generated far fewer Blast colonies as
compared with wild-type ES cells. Furthermore, the expression of Scl
and Gata1 was signficantly lower in Gata2-/- EBs,
consistent with the upregulation of these genes in induced iGATA2 cells
(Fig. 5B). Next, we performed
assays for Blast colonies and primitive erythroid (EryP) cells as well as FACS
analyses for cells expressing endothelial surface markers using iGATA2 ES
cells. In differentiating ES cells, the highest Blast colony number is
typically obtained by replating D2.75 EB cells, as represented by the blue
bars (No Dox/No Dox) in Fig.
5C. We first determined whether induction of GATA2 augments BL-CFC
generation. To this end, Dox was added to differentiating EBs on D2 and EBs
were replated on D2.75 for Blast colony assays. As shown in
Fig. 5C, there was no increase
in Blast colony number (Dox/No Dox). Rather, the number of Blast colonies was
reduced when Dox was added on D2 (Fig.
5C, yellow bars). Even when we replated at earlier time points,
specifically D2.25 and D2.5, we saw no significant increase in the number of
Blast colonies generated (Fig.
5C, yellow bars). We reasoned that the induction of GATA2 from D2
may drive the hemangioblast to rapidly differentiate into its progeny. Thus,
we evaluated the induction of GATA2 at the time of replating. As shown in
Fig. 5C, the Blast colony
number was greatly increased when Dox was added solely to the replating media
(No Dox/Dox). This was true for all EB replating time points, i.e. D2.25,
D2.5, D2.75 and D3 (Fig. 5C,
orange bars). We saw no difference in the percentage of individual Blast
colonies capable of generating both hematopoietic and endothelial cells
between induced and control cells (not shown). Lastly, induction of GATA2 from
D2 and in the replating media (Dox/Dox, green bars in
Fig. 5C) generated an increase
in Blast colonies at early stages (D2.25-D2.5 replating), but resulted in no
change in colony number, as compared with untreated control cells
(Fig. 5C, blue bars), at later
time points (D2.75 and D3 replating). The systemic modulation scheme of
Gata2 expression suggested a true transient nature of the
hemangioblast. If the hemangioblast were truly transient, we reasoned that the
induction of GATA2 from D2 would lead to the precocious emergence of EryP
cells. To this end, D2.75 EBs that had been treated with Dox at D2 were
replated for EryP colonies. Whereas a modest number of EryP colonies could be
generated from untreated D2.75 EBs, GATA2 induction led to a significant
increase in EryP colonies, similar to the number generated from untreated D4
EBs (the peak of EryP colony generation) (compare
Fig. 5D with
Fig. 6B).
|
To further validate this notion, we examined cell proliferation using BrdU
labeling. During EryP formation, cells were recovered from their replating
media by cellulase treatment and grown in the presence of BrdU, with or
without Dox. On average, the BrdU uptake in Dox-treated cells was more than
twice the uptake in untreated EryP cells, indicating greater proliferation in
the presence of exogenous GATA2 (Fig.
6D). In examining previous reports investigating GATA1
(Rylski et al., 2003;
Pan et al., 2005) as well as
our own array data, we discovered that a small subset of cell cycle genes are
relevent to this phenomenon, including the tumor suppressor Rb
(Rb1 - Mouse Genome Informatics), the cyclin complex components
Cdk4 and Ccnd2 (Cyclin D2) and the CDK inhibitors (CDKIs)
p16INK4a, p18INK4c, p19ARF,
p21WAF1 and p27KIP1. These genes show
altered expression dependent upon the status of GATA1
(Rylski et al., 2003;
Pan et al., 2005).
Specifically, the induction of GATA1 results in the suppression of
Cdk4 and Ccnd2 and the activation of the CDKI family members
as well as of Rb. To examine the gene expression of EryP cells upon GATA2
induction, EryP cells were harvested and utilized for qRT-PCR assays. The
results showed that cells with enforced GATA2 had lower expression levels of
the negative regulators Rb and CDKI family members, and increased
expression levels of Cdk4 and Ccnd2
(Fig. 6E). Importantly, this
alteration was unlikely to be due to a change in Gata1 expression, as
the enforced expression of GATA2 led to only a minor suppression of
Gata1 (Fig. 6E).
To examine the endothelial potential of iGATA2 ES cells, D2 EBs were
induced and differentiated for four additional days and subjected to FACS
analyses for endothelial cell surface markers. As shown in
Fig. 7B, induction of GATA2
from D2 resulted in a dramatic increase in Tie2+ cells.
Additionally, GATA2 induction from D2 also led to an increase in the number of
CD31+ as well as of CD31+VE-Cadherin+ cells,
arguing for an increase in endothelial cells generated from D6 EBs
(Fig. 7C,C'). In an
effort to further validate an increase in generation of endothelial cells in
the presence of GATA2, we modified the aortic ring assay
(Nicosia and Ottinetti, 1990a;
Nicosia and Ottinetti, 1990b)
so as to use EBs in place of sections of adult aorta. D6 EBs grown in SR media
with or without Dox from D2 were sandwiched in Matrigel and allowed to sprout
for 6 days in the presence of VEGF. We observed a profound phenotypic variance
in the nature of the sprouts that were generated based upon GATA2 status. In
the absence of GATA2, EBs developed thin, tendril-like sprouting structures
(Fig. 7D,D',G), whereas
EBs grown in the presence of Dox developed flat, heterogeneous-shaped cells
that appeared to be endothelial cells (Fig.
7E,E',G). We sought to verify the nature of these cells, and
tested whether they could take up acetylated low-density lipoproteins, a
characteristic of endothelial cells (Voyta
et al., 1984). The sprouting cells generated from EBs grown in the
presence of Dox were indeed able to take up the Dil-Ac-LDL
(Fig. 7F,F'), whereas the
tendril-like structures from EBs grown the absence of Dox were not
Dil-Ac-LDL+ (not shown). Collectively, these data argue that GATA2
regulates the generation of BL-CFC, hematopoietic and endothelial cells.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Nishikawa et al., 1998;
Faloon et al., 2000;
Chung et al., 2002;
Ema et al., 2003;
Huber et al., 2004). The
present work sought to uncover a molecular signature of the hemangioblast and
to define mechanisms that regulate hemangioblast development. The expression
profile of the Flk-1+Scl+ (hemangioblast) cells derived
from D2.75 EB cells was most similar to the other stem cell populations,
sharing with them the expression of a number of transcription factors as well
as other genes with RNA- and DNA-binding functions. Additionally, the arrays
identified a number of factors preferentially expressed in the hemangioblast.
Certainly, genes that are common with other stem cells could be important for
further determining whether a common pathway exists amongst different stem
cell populations in maintaining `stem-ness'. Conversely, studying genes that
are uniquely expressed in the hemangioblast cell population will be crucial
for understanding how hematopoietic and vascular systems are established.
GATA2 and BMP4 in mesoderm differentiation
The discovery of Gata2 and characterization of its expression
pattern (Minko et al., 2003),
and the knockout phenotype of Gata2-/- animals
(Tsai et al., 1994),
implicated Gata2 as an ideal candidate in molecular control of
hemangioblast development and differentiation. A number of reports have
implicated GATA factors as being downstream of BMP4 signaling, including
Gata2 (Maeno et al.,
1996; Friedle and Knochel,
2002) and Gata4
(Rojas et al., 2005). Here, we
demonstrated that Gata2 was directly induced by BMP4 and that GATA2
activated Bmp4 expression. Although we discovered three conserved
GATA-factor-binding sites in the Bmp4 locus (-1034, -392 and the TGE
site), we showed that GATA2 occupies the TGE site more abundantly than either
the -1034 or -392 sites. Previous work showed that GATA4 and GATA6 utilized
the -392 GATA site and a non-canonical site at -526 to transactivate the
Bmp4 promoter during cardiomyogenesis
(Nemer and Nemer, 2003). Given
the importance of BMP4 in mesodermal differentiation
(Winnier et al., 1995), as
well as the importance of the GATA factors in development, there may be
differential control of Bmp4 in early hematopoietic/endothelial
tissues as compared with cardiomyocytes, and these differences may presumably
account for different mechanisms conferred by GATA2 or GATA4/6, respectively.
Specifically, our studies suggest that GATA2 occupies the TGE site of
Bmp4 and that BMP4 signals in turn activate Gata2
transcription, creating an autoregulatory mesodermal differentiation loop
during early development. By contrast, the -392 (or -526) sites may be bound
by GATA4/6 during later time points of cardiomyogenesis
(Nemer and Nemer, 2003).
Future experiments examining the in vivo occupancy of Bmp4 in
different tissues, as well as loss-of-function studies with candidate
trans-acting factors, will be required to discern the tissue-specific control
of Bmp4 during development.
Our studies indicate that cardiogenic genes including Hand2 and
Nkx2-5 were induced upon enforced GATA2 expression. Moreover, when
EBs were differentiated in the presence of Dox we observed that well over 50%
of EBs, upon prolonged culture, contained foci of beating cardiomyocytes,
whereas no cardiomyocytes were present in the EBs that were formed in the
absence of Dox (not shown). Similarly, the expression of cardiogenic genes
within Flk-1+ cells has recently been reported
(Wang et al., 2006). Whether
the augmentation of cardiogenic genes in iGATA2 cells is the result of direct
trans-activation carried out by GATA2, or is a response to BMP4 signaling, is
unclear and needs to be examined in the future.
|
), and on the current study which showed that GATA2 mediates
Scl expression, we propose that GATA2 expression confers rapid
differentiation of Flk-1+ mesoderm through
Flk-1+Scl+ hemangioblasts into hematopoietic or
endothelial cells by modulating Scl.
We have previously shown that BMP4 alone could generate a modest level of
Scl+ cells and that synergistic signaling by BMP4 and VEGF robustly
generated Scl+ cells (Park et
al., 2004). Here, we have demonstrated that activation of GATA2
robustly yields Scl+ cells. Importantly, activation of GATA2 did
not augment Vegf expression, arguing that this effect was independent
of VEGF signaling. Given the importance of Scl during development as
well as in leukemogenesis, efforts have been invested into trying to elucidate
the mechanism whereby Scl is regulated
(Aplan et al., 1992;
Bockamp et al., 1997;
Gottgens et al., 1997;
Gottgens et al., 2002;
Gottgens et al., 2004). Here,
we have shown in vivo occupancy of the Scl locus by GATA2 and then
GATA1 in a system that recapitulates developing embryos. Only the conserved
GATA site in the promoter region of Scl was occupied by GATA2 or
GATA1 in developing EBs. In our ES/EB model, we did not see either GATA factor
occupying the +19 enhancer site previously posited to confer hematopoietic
specificity to Scl expression in leukemic cells
(Gottgens et al., 2002;
Gottgens et al., 2004). We
argue that during embryogenesis, the +19 enhancer is not occupied, and whether
the GATA occupancy of this enhancer is cell-type-dependent should be
determined in the future. Our data demonstrate that Scl regulation by
GATA2 and GATA1 during ES/EB development is distinctive, such that at the
hemangioblast stage Scl is regulated by GATA2, whereas at the
erythroid stage GATA1 displaces GATA2 to become the dominant GATA factor to
sustain Scl expression. This dynamic occupancy mimics that seen at
the Gata2 locus during erythroid differentiation
(Grass et al., 2003;
Martowicz et al., 2005).
Appreciation of such developmental control of Scl by different GATA
factors should be crucial for further elucidating Scl regulation in
hematopoietic development.
We have demonstrated that GATA2 can function independently within the
primitive erythroid progenitor when induced from D3 and beyond. This induction
of GATA2 within primitive erythroid progenitors increases the number of EryP
cells and the size of EryP colonies. Through BrdU-uptake assays and
examination of cell cycle genes we established that GATA2 confers a
proliferative signal to EryP progenitor cells. The absence of Gata2
during embryogenesis leads to embryonic lethality due to a unique anemia
wherein all fetal hematopoietic organs develop properly yet are entirely
barren of hematopoietic cells (Tsai et
al., 1994). A subsequent study, examining the role that GATA2
plays in the proliferation of primitive hematopoietic cells, showed that a
loss of the tumor suppressor p53 was capable of partially rescuing
the defect associated with Gata2-/- cells
(Tsai and Orkin, 1997). Other
studies of GATA2 have revealed that GATA2 may have discrete functions in
different tissues. Intriguingly, during neural development, GATA2 has been
shown to negatively regulate the cell cycle, allowing for neural
differentiation at the expense of proliferation
(El Wakil et al., 2006).
Additionally, when GATA2 is induced at later stages of hematopoietic
differentiation in the ES-EB system, it is capable of redirecting macrophages
into other hematopoietic cells (Kitajima
et al., 2006). Collectively, GATA2 may have broader,
cell-type-dependent roles in development and differentiation.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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