1: Blood. 2005 Jan 15;105(2):627-34. Epub 2004 Sep 9. 

Hmgb3 deficiency deregulates proliferation and differentiation of common
lymphoid and myeloid progenitors.

Nemeth MJ, Cline AP, Anderson SM, Garrett-Beal LJ, Bodine DM.

Hematopoiesis Section, Genetics and Molecular Biology Branch, National Human
Genome Research Institute, Bethesda, MD 20892-4442, USA

Hmgb3 is an X-linked member of a family of chromatin-binding proteins that is
expressed in primitive hematopoietic cells capable of long-term hematopoietic
repopulation. To examine the role of Hmgb3 in adult hematopoiesis, we generated
Hmgb3-deficient (Hmgb3(-/Y)) mice, which are viable but erythrocythemic.
Hmgb3(-/Y) mice contain normal numbers of hematopoietic stem cells (HSCs), which
generate fewer than normal numbers of common lymphoid progenitors (CLPs) and
common myeloid progenitors (CMPs) and greater than normal numbers of more mature
progenitors. Although fewer Hmgb3(-/Y) primitive progenitor cells are in the
G2/M cell cycle phase, bromodeoxyuridine (BrdU) incorporation demonstrated
enhanced proliferation compared with their wild-type counterparts. Hmgb3(-/Y)
HSCs have increased levels of Gata-2 and c-myb mRNA. We propose that Hmgb3
deficiency leads to a failure of HSCs to expand into normal numbers of CLPs and
CMPs. This defect is compensated for by the ability of Hmgb3(-/Y) progenitors to
expand rapidly and differentiate into normal numbers of hematopoietic cells.

PMID: 15358624 [PubMed - indexed for MEDLINE]


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2: Blood. 2002 Nov 15;100(10):3828-31. Epub 2002 Jul 5. 

Immortalization of yolk sac-derived precursor cells.

Yu WM, Hawley TS, Hawley RG, Qu CK.

Department of Hematopoiesis and the Flow Cytometry Facility, Jerome H. Holland
Laboratory for the Biomedical Sciences, American Red Cross, Rockville, MD 20855,
USA.

Hematopoiesis initiates in the extraembryonic yolk sac. To isolate various types
of precursor cells from this blood cell-forming tissue, yolk sac cells were
immortalized by retroviral-mediated expression of the HOX11 homeobox-containing
gene. Among the cell lines derived, some were able to spontaneously generate
adherent stromal-like cells capable of taking up acetylated low-density
lipoprotein, and they could be induced to form tubelike structures when cultured
on Matrigel. Although these cell lines were negative for hematopoietic cell
surface markers, they gave rise to hematopoietic colonies--containing cells
belonging to the monocytic, megakaryocytic, and definitive erythroid
lineages--when plated in methylcellulose medium supplemented with hematopoietic
growth factors. Low amounts of Flk-1 mRNA could be detected in these cells, and
they showed significant responsiveness to vascular endothelial growth factor,
stem cell factor, basic fibroblast growth factor, and interleukin 6. They also
expressed the transcription factors SCL, GATA2, GATA1, PU.1, and c-myb. These
yolk sac-derived cell lines may represent a transitional stage of early
hematopoietic development.

PMID: 12393673 [PubMed - indexed for MEDLINE]


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3: Exp Hematol. 2002 Aug;30(8):887-95. 

GATA transcription in a small rhodamine 123(low)CD34(+) subpopulation of a
peripheral blood-derived CD34(-)CD105(+) mesenchymal cell line.

Conrad C, Gottgens B, Kinston S, Ellwart J, Huss R.

Institute of Pathology, University of Munich, Germany.

OBJECTIVE: Based on previous animal experiments that suggest the plasticity of
peripheral blood-derived, CD34(-) stem cell lines, the aim of this study was to
isolate CD34(-) stem cell lines from human peripheral blood cells and obtain
evidence of their multipotency and plasticity. MATERIALS AND METHODS: Adherent
growing cells were isolated from peripheral blood mononuclear cells from a
healthy volunteer donor and different cell clones were established after SV40
large-T-antigen-mediated immortalization. The immunophenotype of the cell lines
was investigated by flow cytometry. One particular cell clone, V54/2, was
stained with rhodamine 123, and the Rh123(low) and Rh123(high) subpopulations
were sorted for a reverse transcriptase polymerase chain reaction gene
expression survey and distinct differences in morphology and biologic behavior.
RESULTS: The peripheral blood-derived and fibroblast-like cell line V54/2
expressed high levels of CD10 and CD105 and showed only a very low level
expression of CD34 (<1.0%) and CD117 (c-kit). Among the entire CD34(-)CD105(+)
cell population that transcribed factors such as Myb, Tie-1, and VEGF, there was
a small Rh123(low)CD34(+) subpopulation that transcribed significant levels of
several members of the GATA family of transcription factors. The morphology of
the Rh123(low)CD34(+) (also expressing the P-glycoprotein) was different
compared to the Rh123(high)CD34(-) population. Mesenchymal differentiation into
glial fibrillary acidic protein (GFAP)(+) glial cells could be shown from the
entire CD34(-)CD105(+) cell population. CONCLUSIONS: The findings provide
evidence that it is possible to isolate CD34(-)CD105(+) mesenchymal stem cell
lines from human peripheral blood cells that contain a small subpopulation of
CD34(+) and GATA-transcribing cells. Those cells are potential hematopoietic
progenitors and can be recruited from the CD34(-) stem cell pool. The plasticity
of stem cells seems to require essential molecular tools, such as a panel of
transcription factors, to respond to the environmental demand within a biologic
system.

PMID: 12160840 [PubMed - indexed for MEDLINE]


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4: EMBO J. 2002 Jun 17;21(12):3060-9. 

GATA-2 and GATA-2/ER display opposing activities in the development and
differentiation of blood progenitors.

Kitajima K, Masuhara M, Era T, Enver T, Nakano T.

Department of Molecular Cell Biology, Research Institute for Microbial Diseases,
Osaka University, Suita 565-0871, Japan.

GATA-2 is a zinc finger transcription factor essential for the development of
hematopoiesis. While GATA-2 is generally considered to play an important role in
the biology of hematopoietic stem and progenitor cells, its function within
these compartments is not well understood. Here we have employed both
conditional expression of GATA-2 and conditional activation of a GATA-2/estrogen
receptor (ER) chimera to examine the effect of enforced GATA-2 expression in the
development and differentiation of hematopoietic progenitors from murine
embryonic stem cells. Consistent with the phenotype of GATA-2 null animals,
conditional expression of GATA-2 from a tetracycline-inducible promoter enhanced
the production of hematopoietic progenitors. Conditional activation of a
GATA-2/ER chimera produced essentially opposite effects to those observed with
conditional GATA-2 expression. GATA-2 and GATA-2/ER differ in their binding
activities and transcriptional interactions from other hematopoietic-associated
transcription factors such as c-Myb and PU.1. While we have exploited these
differences in activity to explore the transcriptional networks underlying
hematopoietic cell fate determination, our results suggest that care should be
taken in interpreting results obtained using only chimeric proteins.

PMID: 12065419 [PubMed - indexed for MEDLINE]


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5: J Cell Physiol. 1999 Sep;180(3):390-401. 

Increased expression of the distal, but not of the proximal, Gata1 transcripts
during differentiation of primary erythroid cells.

Vannucchi AM, Linari S, Lin CS, Koury MJ, Bondurant MC, Migliaccio AR.

Division of Hematology, University of Florence and Azienda Ospedale Careggi,
Italy.

Gata1 is expressed from either one of two alternative promoters, the erythroid
(proximal to the AUG) and the testis (distal to the AUG) promoter, both used by
hemopoietic cells. To clarify the role of the distal and proximal Gata1
transcripts in erythroid differentiation, we determined by specific reverse
transcriptase-polymerase chain reactions their relative levels of expression
during the differentiation of erythroid precursors purified from the spleen of
mice treated with phenylhydrazine (PHZ) or infected with the anemia-inducing
strain of the Friend virus (FVA cells). PHZ cells are erythroid precursors that
progress in vivo to erythroblasts in 3 days. Both PHZ and FVA cells
synchronously proliferate and differentiate in vitro in the presence of
erythropoietin (EPO). The levels of total and of distal, but not of proximal,
Gata1 transcripts increased by five- to eightfold during in vivo and in vitro
differentiation of FVA and PHZ cells. The increase in expression was temporally
associated with an increase in the expression of Eklf, Scl, and Nfe2, three
genes required for erythroid differentiation, and preceded by 24 h the
repression of Gata2 and Myb expression. The day 1 PHZ cells that survived 18 h
in the absence of EPO do not express globin genes and express detectable levels
of distal but not of proximal Gata1 transcripts. These cells activate the
expression of the globin genes within 2 h when exposed to EPO. Therefore, during
erythroid differentiation of primary cells, increased expression of distal Gata1
transcripts underlies the increase in the expression of total Gata1 associated
with the establishment of the erythroid differentiation program.

PMID: 10430179 [PubMed - indexed for MEDLINE]


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6: Blood. 1999 Feb 15;93(4):1168-77. 

Expression of alpha4-integrin defines the earliest precursor of hematopoietic
cell lineage diverged from endothelial cells.

Ogawa M, Kizumoto M, Nishikawa S, Fujimoto T, Kodama H, Nishikawa SI.

Department of Molecular Genetics, Faculty of Medicine, Kyoto University, Kyoto,
Japan. mogawa@virus.kyoto-u.ac.jp

Embryonic stem cells can differentiate in vitro into hematopoietic cells through
two intermediate stages; the first being FLK1(+) E-cadherin- proximal lateral
mesoderm and the second being CD45(-) VE-cadherin+ endothelial cells. To further
dissect the CD45(-) VE-cadherin+ cells, we have examined distribution of
alpha4-integrin on this cell population, because alpha4-integrin is the molecule
expressed on hematopoietic stem cells. During culture of FLK1(+) E-cadherin-
cells, CD45(-) VE-cadherin+ alpha4-integrin- cells differentiate first, followed
by alpha4-integrin+ cells appearing in both CD45(-) VE-cadherin+ and CD45(-)
VE-cadherin- cell populations. In the CD45(-) VE-cadherin+ cell population,
alpha4-integrin+ subset but not alpha4-integrin- subset had the potential to
differentiate to hematopoietic lineage cells, whereas endothelial cell
progenitors were present in both subsets. The CD45(-) VE-cadherin-
alpha4-integrin+ cells also showed hematopoietic potential. Reverse
transcription-polymerase chain reaction analyses showed that differential
expression of the Gata2 and Myb genes correlated with the potential of the
alpha4-integrin+ cells to give rise to hematopoietic cell differentiation.
Hematopoietic CD45(-) VE-cadherin+ alpha4-integrin+ cells were also present in
the yolk sac and embryonic body proper of 9.5 day postcoitum mouse embryos. Our
results suggest that the expression of alpha4-integrin is a marker of the
earliest precursor of hematopoietic cell lineage that was diverged from
endothelial progenitors.

PMID: 9949159 [PubMed - indexed for MEDLINE]


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7: Development. 1996 Dec;122(12):3839-50. 

Regulation and function of transcription factor GATA-1 during red blood cell
differentiation.

Briegel K, Bartunek P, Stengl G, Lim KC, Beug H, Engel JD, Zenke M.

Max-Delbruck-Center for Molecular Medicine, MDC, Berlin, Germany.

The tissue-specific transcription factor GATA-1 is a key regulator of red blood
cell differentiation. One seemingly contradictory aspect of GATA-1 function is
that, while it is abundant in erythroid progenitor cells prior to the onset of
overt differentiation, it does not significantly activate known GATA-1 target
genes in those cells. To investigate the mechanisms underlying GATA-1 function
during the transition from early to late erythropoiesis, we have examined its
expression and activity in normal avian erythroid progenitor cells before and
after induction of differentiation. In these primary progenitor cells, GATA-1
protein was predominantly located in the cytoplasm, while induction of
differentiation caused its rapid relocalization to the nucleus, suggesting that
nuclear translocation constitutes an important regulatory step in GATA-1
activation. As an alternative way of addressing the same question, we also
ectopically expressed a GATA-1/estrogen receptor fusion protein (GATA-1/ER) in
red blood cell progenitors, where nuclear translocation of, and transcriptional
activation by, this hybrid factor are conditionally controlled by estrogen. We
found that hormone-activated GATA-1/ER protein accelerated red blood cell
differentiation, and concomitantly suppressed cell proliferation. These
phenotypic effects were accompanied by a simultaneous suppression of c-myb and
GATA-2 transcription, two genes thought to be involved in the proliferative
capacity of hematopoietic progenitor cells. Thus, GATA-1 appears to promote
differentiation in committed erythroid progenitor cells both by inducing
differentiation-specific genes and by simultaneously suppressing genes involved
in cell proliferation.

PMID: 9012505 [PubMed - indexed for MEDLINE]


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8: Proc Natl Acad Sci U S A. 1996 May 28;93(11):5313-8. 

The transcription factors c-myb and GATA-2 act independently in the regulation
of normal hematopoiesis.

Melotti P, Calabretta B.

Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas
Jefferson University, Philadelphia, PA 19107, USA.

The transcription factors c-myb and GATA-2 are both required for blood cell
development in vivo and in vitro. However, very little is known on their
mechanism(s) of action and whether they impact on complementary or overlapping
pathways of hematopoietic proliferation and differentiation. We report here that
embryonic stem (ES) cells transfected with c-myb or GATA-2 cDNAs, individually
or in combination, underwent hematopoietic commitment and differentiation in the
absence of added hematopoietic growth factors but that stimulation with c-kit
and flt-3 ligands enhanced colony formation only in the c-myb transfectants.
This enhancement correlated with c-kit and flt-3 surface receptor up-regulation
in c-myb-(but not GATA-2-) transfected ES cells. Transfection of ES cells with
either a c-myb or a GATA-2 antisense construct abrogated erythromyeloid
colony-forming ability in methyl cellulose; however, introduction of a
full-length GATA-2 or c-myb cDNA, respectively, rescued the
hematopoiesis-deficient phenotype, although only c-myb-rescued ES cells
expressed c-kit and flt-3 surface receptors and formed increased numbers of
hematopoietic colonies upon stimulation with the cognate ligands. These results
are in agreement with previous studies indicating a fundamental role of c-myb
and GATA-2 in hematopoiesis. Of greater importance, our studies suggest that
GATA-2 and c-myb exert their roles in hematopoietic gene regulation through
distinct mechanisms of action in nonoverlapping pathways.

PMID: 8643572 [PubMed - indexed for MEDLINE]


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9: Proc Natl Acad Sci U S A. 1995 May 9;92(10):4601-5. 

Pluripotent hematopoietic stem cells contain high levels of mRNA for c-kit,
GATA-2, p45 NF-E2, and c-myb and low levels or no mRNA for c-fms and the
receptors for granulocyte colony-stimulating factor and interleukins 5 and 7.

Orlic D, Anderson S, Biesecker LG, Sorrentino BP, Bodine DM.

Hematopoiesis Section, National Center for Human Genome Research, National
Institutes of Health, Bethesda, MD 20892, USA.

Pluripotent hematopoietic stem cells (PHSCs) were highly enriched from mouse
bone marrow by counterflow centrifugal elutriation, lineage subtraction, and
fluorescence-activated cell sorting based on high c-kit receptor expression
(c-kitBR). We used reverse transcriptase polymerase chain reaction to assay the
c-kitBR subset and the subsets expressing low (c-kitDULL) and no (c-kitNEG)
c-kit receptor for expression of mRNA encoding hematopoietic growth factor
receptors and transcription factors. The c-kitBR cells had approximately
3.5-fold more c-kit mRNA than unfractionated bone marrow cells. The c-kitDULL
cells had 47-58% of the c-kit mRNA found in c-kitBR cells and the c-kitNEG cells
had 4-9% of the c-kit mRNA present in c-kitBR cells. By comparing mRNA levels in
c-kitBR cells (enriched for PHSCs) with those of unfractionated bone marrow, we
demonstrated that c-kitBR cells contained low or undetectable levels of mRNA for
c-fms, granulocyte colony-stimulating factor receptor, interleukin 5 receptor
(IL-5R), and IL-7R. These same cells had moderate levels of mRNA for
erythropoietin receptor, IL-3R subunits IL-3R alpha (SUT-1), AIC-2A, and AIC-2B,
IL-6R and its partner gp-130, and the transcription factor GATA-1 and high
levels of mRNA for transcription factors GATA-2, p45 NF-E2, and c-myb. We
conclude from these findings that PHSCs are programmed to interact with stem
cell factor, IL-3, and IL-6 but not with granulocyte or macrophage
colony-stimulating factor. These findings also indicate that GATA-2, p45 NF-E2,
and c-myb activities may be involved in PHSC maintenance or proliferation.

PMID: 7538677 [PubMed - indexed for MEDLINE]


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10: Genes Dev. 1994 May 15;8(10):1184-97. 

Novel insights into erythroid development revealed through in vitro
differentiation of GATA-1 embryonic stem cells.

Weiss MJ, Keller G, Orkin SH.

Division of Hematology-Oncology, Children's Hospital, Dana Farber Cancer
Institute, Boston, Massachusetts.

Mouse embryonic stem (ES) cells lacking the transcription factor GATA-1 do not
produce mature red blood cells either in vivo or in vitro. To define the
consequences of GATA-1 loss more precisely, we used an in vitro ES cell
differentiation assay that permits enumeration of primitive (EryP) and
definitive (EryD) erythroid precursors and recovery of pure erythroid colonies.
In contrast to normal ES cells, GATA-1- ES cells fail to generate EryP
precursors. EryD precursors, however, are normal in number but undergo
developmental arrest and death at the proerythroblast stage. Contrary to initial
expectations, arrested GATA-1(-)-definitive proerythroblasts express GATA target
genes at normal levels. Transcripts of the related factor GATA-2 are remarkably
elevated in GATA-1- proerythroblasts. These findings imply substantial
interchangeability of GATA factors in vivo and suggest that GATA-1 normally
serves to repress GATA-2 during erythropoiesis. The approach used here is a
paradigm for the phenotypic analysis of targeted mutations affecting
hematopoietic development.

PMID: 7926723 [PubMed - indexed for MEDLINE]


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