1: Blood. 2005 Aug 1;106(3):860-70. Epub 2005 Apr 14. 

Hematopoietic differentiation of human embryonic stem cells progresses through
sequential hematoendothelial, primitive, and definitive stages resembling human
yolk sac development.

Zambidis ET, Peault B, Park TS, Bunz F, Civin CI.

Division of Immunology and Hematopoiesis, Sidney Kimmel comprehensive Cancer
Center at John Hokins, The John Hopkins School of Medicine, Baltimore, MD, USA.
ezambid1@jhmi.edu

We elucidate the cellular and molecular kinetics of the stepwise differentiation
of human embryonic stem cells (hESCs) to primitive and definitive
erythromyelopoiesis from human embryoid bodies (hEBs) in serum-free clonogenic
assays. Hematopoiesis initiates from CD45 hEB cells with emergence of
semiadherent mesodermal-hematoendothelial (MHE) colonies that can generate
endothelium and form organized, yolk sac-like structures that secondarily
generate multipotent primitive hematopoietic stem progenitor cells (HSPCs),
erythroblasts, and CD13+CD45+ macrophages. A first wave of hematopoiesis follows
MHE colony emergence and is predominated by primitive erythropoiesis
characterized by a brilliant red hemoglobinization, CD71/CD325a (glycophorin A)
expression, and exclusively embryonic/fetal hemoglobin expression. A second wave
of definitive-type erythroid burst-forming units (BFU-e's), erythroid
colony-forming units (CFU-e's), granulocyte-macrophage colony-forming cells
(GM-CFCs), and multilineage CFCs follows next from hEB progenitors. These stages
of hematopoiesis proceed spontaneously from hEB-derived cells without
requirement for supplemental growth factors during hEB differentiation. Gene
expression analysis of differentiating hEBs revealed that initiation of
hematopoiesis correlated with increased levels of SCL/TAL1, GATA1, GATA2, CD34,
CD31, and the homeobox gene-regulating factor CDX4 These data indicate that
hematopoietic differentiation of hESCs models the earliest events of embryonic
and definitive hematopoiesis in a manner resembling human yolk sac development,
thus providing a valuable tool for dissecting the earliest events in human HSPC
genesis.

PMID: 15831705 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


2: Blood. 2005 Jul 15;106(2):477-84. Epub 2005 Apr 5. 

Haploinsufficiency of GATA-2 perturbs adult hematopoietic stem-cell homeostasis.

Rodrigues NP, Janzen V, Forkert R, Dombkowski DM, Boyd AS, Orkin SH, Enver T,
Vyas P, Scadden DT.

Center for Regenerative Medicine and Technology, Massachusetts General Hospital,
Boston, MA 02129, USA.

The zinc finger transcription factor GATA-2 plays a fundamental role in
generating hematopoietic stem-cells in mammalian development. Less well defined
is whether GATA-2 participates in adult stem-cell regulation, an issue we
addressed using GATA-2 heterozygote mice that express reduced levels of GATA-2
in hematopoietic cells. While GATA-2+/- mice demonstrated decreases in some
colony-forming progenitors, the most prominent changes were observed within the
stem-cell compartment. Heterozygote bone marrow had a lower abundance of
Lin(-)c-kit(+)Sca-1(+)CD34- cells and performed poorly in competitive
transplantation and quantitative week-5 cobblestone area-forming cell (CAFC)
assays. Furthermore, a stem-cell-enriched population from GATA1+/- marrow was
more quiescent and exhibited a greater frequency of apoptotic cells associated
with decreased expression of the anti-apoptotic gene Bcl-xL. Yet the
self-renewal potential of the +/- stem-cell compartment, as judged by serial
transplantations, was unchanged. These data indicate compromised primitive cell
proliferation and survival in the setting of a lower GATA-2 gene dose without a
change in the differentiation or self-renewal capacity of the stem-cells that
remain. Thus, GATA-2 dose regulates adult stem-cell homeostasis by affecting
select aspects of stem cell function.

PMID: 15811962 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


3: Dev Cell. 2005 Jan;8(1):109-16. 

Loss of gata1 but not gata2 converts erythropoiesis to myelopoiesis in zebrafish
embryos.

Galloway JL, Wingert RA, Thisse C, Thisse B, Zon LI.

Stem Cell Program and Division of Hematology/Oncology, Children's Hospital
Boston, Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard
Medical School, Boston, MA 02115, USA.

The differentiation of hematopoietic progenitors into erythroid or myeloid cell
lineages is thought to depend upon relative levels of the transcription factors
gata1 and pu.1. While loss-of-function analysis shows that gata1 is necessary
for terminal erythroid differentiation, no study has demonstrated that loss of
gata1 alters myeloid differentiation during ontogeny. Here we provide in vivo
evidence that loss of Gata1, but not Gata2, transforms primitive blood
precursors into myeloid cells, resulting in a massive expansion of granulocytic
neutrophils and macrophages at the expense of red blood cells. In addition to
this fate change, expression of many erythroid genes was found to be
differentially dependent on Gata1 alone, on both Gata1 and Gata2, or independent
of both Gata factors, suggesting that multiple pathways regulate erythroid gene
expression. Our studies establish a transcriptional hierarchy of Gata factor
dependence during hematopoiesis and demonstrate that gata1 plays an integral
role in directing myelo-erythroid lineage fate decisions during embryogenesis.

PMID: 15621534 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


4: Leuk Res. 2004 Nov;28(11):1227-37. 

Analysis of the relationship between Scl transcription factor complex protein
expression patterns and the effects of LiCl on ATRA-induced differentiation in
blast cells from patients with acute myeloid leukemia.

Rice AM, Holtz KM, Karp J, Rollins S, Sartorelli AC.

Department of Pharmacology, Cancer Center, Yale University School of Medicine,
333 Cedar Street, New Haven, CT 06520, USA.

Exogenous expression of the transcription factor Scl (Tal1) in WEHI-3B D+
myelomonocytic leukemia cells interferes with their capacity to respond to
all-trans retinoic acid (ATRA) induced differentiation; combination of ATRA with
LiCl, however, circumvents the inhibition of differentiation produced by Scl. To
gain information on the possible involvement of this transcription factor in the
non-responsiveness of acute myelocytic leukemia (AML) patients to ATRA, we
compared the endogenous expression levels of Scl and its transcription complex
partners [i.e., Rbtn1 (LMO1), Rbtn2 (LMO2), Ldb1, and GATA family proteins] in
leukemic blast cells from patients with AML and acute promyelocytic leukemia
(APL), and determined the effects of lithium chloride alone or in combination
with ATRA on the capacity of blast cells to differentiate during short-term ex
vivo culture. Levels of Scl, Rbtn2, GATA1, and Ldb1 expression were comparable
in AML and APL blasts, while the levels of expression of Rbtn1, GATA2, and GATA3
were absent or markedly lower in APL cells. Differentiation markers (cell
surface myeloid antigens CD11b, CD15, CD14, and CD33) were also analyzed in
blast cells. ATRA produced changes in at least one surface antigen
differentiation marker in 89% of patient blasts, while LiCl caused such changes
in 72% of the leukemic cells of patients. The combination of LiCl and ATRA
induced the differentiation of leukemic blasts from 94% of patients. Although
the expression of the transcription factors did not act as individual predictors
of responsiveness or non-responsiveness to the inducers of differentiation, ATRA
or ATRA plus LiCl, the addition of LiCl to ATRA increased the differentiation
response over that of ATRA alone in a number of leukemic samples. These findings
suggest that the combination of LiCl and ATRA may produce some clinical benefit
in the treatment of the myeloid leukemias.

PMID: 15380350 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


5: Hum Cell. 2004 Jun;17(2):85-92. 

Transcription factor expression in cell lines derived from natural killer-cell
and natural killer-like T-cell leukemia-lymphoma.

Matsuo Y, Drexler HG, Harashima A, Okochi A, Shimizu N, Orita K.

Fujisaki Cell Center, Hayashibara Biochemical Labs, Okayama 702-8006, Japan.
yomatsuo@hayashibara.co.jp

Although a number of transcription factors (TFs) have been identified that play
a pivotal role in the development of hematopoietic lineages, only little is
known about factors that may influence development and lineage commitment of
natural killer (NK) or NK-like T (NKT)-cells. Obviously to fully appreciate the
NK- and NKT-cell differentiation process, it is important to identify and
characterize the TFs effecting the NK- and NKT-cell lineage. Furthermore, these
TFs may play a role in NK- or NKT-cell leukemias, in which the normal
differentiation program is presumably disturbed. The present study analyzed the
expression of the following 13 TFs: AML1, CEBPA, E2A, ETS1, GATA1, GATA2, GATA3,
IKAROS, IRF1, PAX5, PU1, TBET and TCF1 in 7 malignant NK-cell lines together
with 5 malignant NKT-cell lines, 5 T-cell acute lymphoblastic leukemia (ALL)
cell lines including 3 gamma/delta T-cell receptor (TCR) type and 2 alpha/beta
TCR type, and 3 B-cell precursor (BCP) leukemia cell lines. AML1, E2A, ETS1,
IKAROS and IRF1 were found to be positive for all cell lines tested whereas
GATA1 turned out to be universally negative. CEBPA, PAX5 and PU1 were negative
for all cell lines tested except in the three positive BCP-cell lines. GATA2 was
positive for 3/5 T-cell lines but negative for the other cell lines. GATA3 was
positive for 7/7 NK-, 4/5 NKT-, 5/5 T- and 2/3 BCP-cell lines. TBET was positive
for all NK- and NKT-cell lines and negative for all T- and BCP-cell lines except
one BCP-cell line. In contrast to the expression of TBET, TCF1 was negative for
all NK- and NKT-cell lines, being positive for 4/5 T- and 1/3 BCP-cell lines.
Expression analysis of TFs revealed that NK- and NKT-cell lines showed identical
profiles, clearly distinct from those of the other T-ALL or BCP-ALL
leukemia-derived cell lines..

PMID: 15369140 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


6: Genes Chromosomes Cancer. 2004 Jul;40(3):179-89. 

Molecular heterogeneity in AML/MDS patients with 3q21q26 rearrangements.

Lahortiga I, Vazquez I, Agirre X, Larrayoz MJ, Vizmanos JL, Gozzetti A, Calasanz
MJ, Odero MD.

Department of Genetics, University of Navarra, Pamplona, Spain.

Patients with 3q21q26 rearrangements seem to share similar clinicopathologic
features and a common molecular mechanism, leading to myelodysplasia or acute
myeloid leukemia (AML). The ectopic expression of EVI1 (3q26) has been
implicated in the dysplasia that characterizes this subset of myeloid
neoplasias. However, lack of EVI1 expression has been reported in several cases,
and overexpression of EVI1 was detected in 9% of AML cases without 3q26
abnormalities. We report the molecular characterization of seven patients with
inv(3)(q21q26), t(3;3)(q21;q26) or related abnormalities. EVI1 expression was
detected in only one case, and thus ectopic expression of this gene failed to
explain all of these cases. GATA2 (3q21) was found to be overexpressed in 5 of
the 7 patients. GATA2 is highly expressed in stem cells, and its expression
dramatically decreases when erythroid and megakaryocytic differentiation
proceeds. No mutations in GATA1 were found in any patient, excluding loss of
function of GATA1 as the cause of GATA2 overexpression. We report finding
molecular heterogeneity in patients with 3q21q26 rearrangements in both
breakpoints and in the expression pattern of the genes near these breakpoints.
Our data suggest that a unique mechanism is not likely to be involved in 3q21q26
rearrangements. Copyright 2004 Wiley-Liss, Inc.

PMID: 15138998 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


7: EMBO J. 2003 Aug 1;22(15):3887-97. 

Myeloid lineage switch of Pax5 mutant but not wild-type B cell progenitors by
C/EBPalpha and GATA factors.

Heavey B, Charalambous C, Cobaleda C, Busslinger M.

Research Institute of Molecular Pathology, Vienna Biocenter,Dr. Bohr-Gasse 7,
A-1030 Vienna, Austria.

The developmental potential of hematopoietic progenitors is restricted early on
to either the erythromyeloid or lymphoid lineages. The broad developmental
potential of Pax5(-/-) pro-B cells is in apparent conflict with such a strict
separation, although these progenitors realize the myeloid and erythroid
potential with lower efficiency compared to the lymphoid cell fates. Here we
demonstrate that ectopic expression of the transcription factors C/EBPalpha,
GATA1, GATA2 and GATA3 strongly promoted in vitro macrophage differentiation and
myeloid colony formation of Pax5(-/-) pro-B cells. GATA2 and GATA3 expression
also resulted in efficient engraftment and myeloid development of Pax5(-/-)
pro-B cells in vivo. The myeloid transdifferentiation of Pax5(-/-) pro-B cells
was accompanied by the rapid activation of myeloid genes and concomitant
repression of B-lymphoid genes by C/EBPalpha and GATA factors. These data
identify the Pax5(-/-) pro-B cells as lymphoid progenitors with a latent myeloid
potential that can be efficiently activated by myeloid transcription factors.
The same regulators were unable to induce a myeloid lineage switch in Pax5(+/+)
pro-B cells, indicating that Pax5 dominates over myeloid transcription factors
in B-lymphocytes.

PMID: 12881423 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


8: Exp Cell Res. 2003 May 1;285(2):258-67. 

5-azacytidine reactivates the erythroid differentiation potential of the
myeloid-restricted murine cell line 32D Ro.

Baiocchi M, Di Rico C, Di Pietro R, Di Baldassarre A, Migliaccio AR.

Department of Hematology, Instituto Superiore Sanita, Rome, Italy.

32D cells grown for 1 year in interleukin-3 (IL-3) and granulocyte
colony-stimulating factor (G-CSF) generated the 32D Ro cell line which retained
the parental mast cell phenotype but lost ability to generate erythroid cells in
response to erythropoietin (EPO). In order to clarify the mechanisms underlying
such restriction, we compared 32D and 32D Ro cells for their capacity to express
erythroid-specific transcription factors (Gata1, Gata2, Scl, Nef2, Eklf, and Id)
and the capacity of short exposure to 5-azacytidine (5-AzaC) to reactivate
erythroid differentiation potential in 32D Ro cells. By Northern analysis, the
two cell lines expressed similar levels of all these genes. However, after being
treated with 5-AzaC, 32D Ro cells acquired the ability to generate EPO-dependent
clones (1 clone/10(4) cells) which gave rise to EPO-dependent cell lines. All
the 10 EPO-responsive cell lines independently isolated from 5-AzaC-treated 32D
Ro cells had erythroid morphology and expressed high levels of alpha- and
beta-globin. In contrast, none of the IL-3-dependent and granulocyte/macrophage
colony-stimulating factor-dependent clones concurrently isolated, as a control,
showed erythroid properties. Therefore, 5-AzaC treatment reactivates the
potential of the myeloid-restricted 32D Ro cells to generate EPO-responsive
erythroid clones suggesting that gene methylation played an important role in
the G-CSF-mediated restriction/activation of the differentiation potential of
these cells.

PMID: 12706120 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


9: Hum Genet. 2003 Jan;112(1):42-9. Epub 2002 Oct 16. 

Molecular cloning and characterization of the GATA1 cofactor human FOG1 and
assessment of its binding to GATA1 proteins carrying D218 substitutions.

Freson K, Thys C, Wittewrongel C, Vermylen J, Hoylaerts MF, Van Geet C.

Center for Molecular and Vascular Biology, University of Leuven,
UZ-Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium.

Erythroid and megakaryocytic lineage differentiation and maturation are
regulated via cooperation between transcription factor GATA1 and its essential
cofactor friend-of-GATA1 (FOG1). The interaction between these two murine
proteins is well studied in vitro and depends on the binding of Fog1 to the
N-terminal zinc finger (N-finger) of Gata1. We identified the human FOG1 gene on
chromosome 16q24 and found expression mainly in hematopoietic cells and also in
several other tissues. Sequencing of FOG1 cDNA revealed a 1006 amino-acid
protein that contained nine zinc fingers, highly homologous to murine Fog1
fingers. The amino acid sequence and the GATA1-binding capacity of the human and
murine finger 5 are however different. Ex vivo binding studies demonstrated that
FOG1 interacts with both GATA1 and GATA2. We and others have described patients
with mutations in the GATA1 N-finger (V205 M, D218G, D218Y, or G208S), who
suffer from macrothrombocytopenia and erythrocyte abnormalities. We now show ex
vivo that the interaction between GATA1 and FOG1 is indeed disturbed in
platelets and erythrocytes of those patients carrying D218 GATA1 mutations. The
identification of the human FOG1 gene will enable the genetic screening of
patients with non X-linked thrombocytopenia and dyserythropoiesis.

PMID: 12483298 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


10: Development. 2002 Nov;129(21):5093-101. 

HrT is required for cardiovascular development in zebrafish.

Szeto DP, Griffin KJ, Kimelman D.

Department of Biochemistry, Box 357350, University of Washington, Seattle
98195-7530, USA.

The recently identified zebrafish T-box gene hrT is expressed in the developing
heart and in the endothelial cells forming the dorsal aorta. Orthologs of hrT
are expressed in cardiovascular cells from Drosophila to mouse, suggesting that
the function of hrT is evolutionarily conserved. The role of hrT in
cardiovascular development, however, has not thus far been determined in any
animal model. Using morpholino antisense oligonucleotides, we show that
zebrafish embryos lacking hrT function have dysmorphic hearts and an absence of
blood circulation. Although the early events in heart formation were normal in
hrT morphant embryos, subsequently the hearts failed to undergo looping, and
late onset defects in chamber morphology and gene expression were observed. In
particular, we found that the loss of hrT function led to a dramatic
upregulation of tbx5, a gene required for normal heart morphogenesis.
Conversely, we show that overexpression of hrT causes a significant
downregulation of tbx5, indicating that one key role of hrT is to regulate the
levels of tbx5. Secondly, we found that HrT is required to inhibit the
expression of the blood lineage markers gata1 and gata2 in the most posterior
lateral plate mesoderm. Finally, we show that HrT is required for vasculogenesis
in the trunk, leading to similar vascular defects to those observed in midline
mutants such as floating head. hrT expression in the vascular progenitors
depends upon midline mesoderm, indicating that this expression is one important
component of the response to a midline-derived signal during vascular
morphogenesis.

PMID: 12397116 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


11: 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]


---------------------------------------------------------------


12: 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]


---------------------------------------------------------------