First published online September 1, 2004
doi: 10.1242/10.1242/dev.01310
Development 131, 4623-4634 (2004)
Published by The Company of Biologists 2004
Hypoxia affects mesoderm and enhances hemangioblast specification during early development
Diana L. Ramírez-Bergeron1,2,
Anja Runge1,2,
Karen D. Cowden Dahl2,
Hans Joerg Fehling3,
Gordon Keller4 and
M. Celeste Simon1,2,*
1 Howard Hughes Medical Institute and Abramson Family Cancer Research Institute,
University of Pennsylvania School of Medicine, Philadelphia, PA 19104,
USA
2 Abramson Family Cancer Research Institute, University of Pennsylvania School
of Medicine, Philadelphia, PA 19104, USA
3 Department of Immunology, Medical Faculty/University Clinics, 89070 Ulm,
Germany
4 Carl C, Icahn Center for Gene Therapy and Molecular Medicine, Mount Sinai
School of Medicine, New York, NY 10029, USA
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Fig. 1. Characterization of methylcellulose colonies. (A) Micrographs of
representative colonies from methylcellulose cultures: 2° EB colony (A
'), transitional colony (Trans-CFC; D') and hemangioblast colony
(BL-CFC; G'). Microwells of colonies replated on matrigel: 2° EB
with adherent mesodermal cell type (B',C'); two independent
transitional colonies showing mostly hematopoietic cells (E',F');
BL-CFCs showing both adherent endothelial cells (arrows) and overlaying
non-adherent hematopoietic cells (arrowhead; H',I'). (B) Single
hemangioblasts were replated on matrigel for one week and treated with
fluorescent DiI-Ac-LDL. Phase contrast (left panel) and fluorescent micrograph
(right panel) images are shown. (C) RNA from individual
Arnt+/+ and Arnt–/– 2°
EB, Trans-CFC and BL-CFC colonies were analyzed for the expression of the
indicated genes by radioactive RT-PCR: ß-actin (control), Rex1
(ES cells), Bry (mesoderm), Scl (endothelial/hematopoietic
cells) and GATA1 (hematopoietic cells). As BL-CFCs are generally smaller
colonies, the signal for ß-actin is lower.
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Fig. 2. ARNT is required for the appropriate development of hemangioblasts, and
hypoxia accelerates the kinetics of their appearance. ES cell lines were
differentiated into EBs and replated in a BL-CFC methylcellulose assay. (A)
Five independent Arnt–/– clones (patterned
bars) were differentiated for 2.75 days at 21% O2, replated and
compared with the wild-type control (black bar). (B)
Arnt+/+ (left panels) and
Arnt–/– (right panels) ES lines were
differentiated into EBs under normoxic (white bars, N; 21% O2) or
hypoxic (black bars, H; 3% O2) conditions, for the indicated times,
and replated for hemangioblast analysis. The number of transitional colonies
is stimulated by hypoxia in Arnt+/+ cultures (days 2 and
2.5), but mutants are not affected (upper panels). Hypoxia also accelerates
the kinetics and increases the number of BL-CFCs for wild-type cells but not
for Arnt–/– clones (lower panels). Error bars
represent the standard error of the mean (s.e.m.) for triplicate assays. (C)
Kinetics of Arnt+/+ BL-CFC formation from 2 to 3.5 days,
comparing normoxic (blue) and hypoxic (red) conditions.
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Fig. 5. VEGF is not necessary for FLK1+ cell production, but is
necessary for the generation of BL-CFCs. (A) Arnt+/+,
Arnt–/–, Vegf+/– and
Vegf–/– ES clones were differentiated for 3 days
under normoxic (21% O2) or hypoxic (3% O2) conditions
and replated for their hemangioblast potential. Error bars represent SEMs of
triplicate cultures. (B) Generation of BL-CFCs in Arnt+/+
cultures is inducible with the addition of VEGF (5 and 10 ng/ml) during early
time points (day 1.5-2.5), but exogenous VEGF does not rescue the
Arnt–/– cultures. (C) VEGF and bFGF do not
rescue FLK1+ cell production in
Arnt–/– EB cultures;
Vegf+/– and Vegf–/– cells
exhibit no FLK1+ deficiency and FLK1+ cell numbers can
be further induced with the addition of VEGF and bFGF, or by hypoxia
treatment.
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Fig. 3. Hypoxia influences the kinetics of mesoderm differentiation. Flow cytometry
was performed on dissociated cells obtained from EBs harvested at the
indicated time points. (A) The number of FLK1+ cells detected in
differentiated Arnt+/+ EBs is increased by hypoxic
conditions. In direct contrast, the number of FLK1+ cells in two
independent Arnt–/– cell lines is reduced in
normoxia and is not enhanced by 3% O2. (B) Hypoxia stimulates the
production of GFP+ cells in GFP-BRY ES cells as early as day 2 of
differentiation.
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Fig. 4. Hypoxia influences the expression of genes associated with mesoderm
differentiation. RT-PCR assays performed on RNA isolated from normoxic or
hypoxic undifferentiated (ES) and EB cultures. (A) RT-PCR analyses of
Arnt+/+ cells and two independent
Arnt–/– lines differentiated under normoxic
(N, 21%) or hypoxic (H, 3%) conditions. ß-actin serves as a loading
control. (B) Relative levels of gene expression from
Arnt+/+ and Arnt–/–
cultures under normoxic (N, 21%) or hypoxic (H, 3%) conditions assayed by
real-time detection (RTD)-PCR. Transcripts are normalized to 18S RNA.
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Fig. 6. The BL-CFC defect in Arnt–/– cells is cell
extrinsic. (A) Arnt+/+ neos
lacZ– and Arnt–/–
neor lacZ+ ES cells co-cultured during EB
differentiation, dissociated and replated in methylcellulose with or without
G418. (B) Under G418 selection, all surviving BL-CFCs stained blue indicating
ß-galactosidase activity from Arnt–/–
neor lacZ+ cells. Wild-type clones only
survived in the absence of G418.
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Fig. 7. Model of ARNT activity during early embryonic development. HIF responses
are required for the proper differentiation, survival or proliferation of
mesoderm and hemangioblasts. In the absence of ARNT, mesoderm differentiation
is delayed. Furthermore, ARNT deficiency results in reduced mesoderm
differentiation and increased transitional cell numbers, suggesting a block in
commitment to the hemangioblast phenotype. Fewer hemangioblasts lead to a
reduction in the numbers of hematopoietic and endothelial precursors.
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© The Company of Biologists Ltd 2004
