First published online January 16, 2004
doi: 10.1242/10.1242/dev.00968
Development 131, 693-702 (2004)
Published by The Company of Biologists 2004
SCL interacts with VEGF to suppress apoptosis at the onset of hematopoiesis
Richard Martin1,2,
Rachid Lahlil1,
Annette Damert3,*,
Lucile Miquerol3,
,
Andras Nagy3,
Gordon Keller4 and
Trang Hoang1,2,5,
,
1 Laboratory of Hematopoiesis and Leukemia, Clinical Research Institute of
Montreal, Montreal, Canada
2 Department of Medicine, Division of Experimental Medicine, McGill University,
Montreal, Canada
3 Mount Sinai Hospital, Samuel Lunenfeld Research Institute, Toronto,
Canada
4 Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of
Medicine, New York, USA
5 Departments of Pharmacology, Biochemistry and Molecular Biology, University of
Montreal, Montreal, Canada
View larger version (63K):
[in a new window]
|
Fig. 1. VEGF stimulates primitive erythropoiesis. (A) ES cells were differentiated
into EBs in either the presence or absence of VEGF, which was added on day 3
(d3). The hematopoietic precursor content of EBs was assessed by plating
dissociated cells in the presence of hematopoietic growth factors (See
Materials and methods). LIF, leukemia inhibitory factor; EryP,
primitive erythroid colonies. (B-E) Morphology of day-7 EBs.
Vegf–/– ES cells were differentiated for 7
days in either the absence (B,C) or presence (D,E) of VEGF (5 ng
ml–1). Note the larger size and the intensity of
hemoglobinization in VEGF-treated EBs. (F) EB size was estimated by
integrating individual surface area using Northern Eclipse software.
Histograms illustrate the distribution of individual EB area. The range
covered by columns is equivalent and determined arbitrarily. Data were
analyzed by Student's t-test: –VEGF, n=102; +VEGF,
n=44; P<0.001. (G) VEGF stimulates a dose-dependent increase
in the number of primitive erythroid progenitors.
Vegf–/– ES cells were differentiated in the
presence of increasing concentrations of VEGF and assayed for primitive
erythroid precursors (Materials and methods). Results are the mean±s.d.
of duplicates and are representative of five independent experiments. (H)
Morphology of day 4 EryP. (I) Morphology of colony cells revealed
by Wright-Giesma stain. Scale bars: 100 µm in B-E,H; 10 µm in I.
|
|
View larger version (52K):
[in a new window]
|
Fig. 2. Clonal analysis of the effect of VEGF during primitive erythropoiesis. (A)
VEGF increases the number of EryP per single EB. On day 7 of
culture either with or without VEGF (5 ng ml–1), 25 EBs were
picked at random and assayed individually into hematopoiesis. Histograms
represent the frequency of EBs giving rise to the indicated number of
EryP. (B) Gene-expression analysis of day-7 EBs treated with VEGF.
Individual EBs (10) were dissociated as above and analyzed for
hematopoietic-marker expression. No reverse transcriptase (RT) served as a
control for genomic DNA contamination. Membranes were hybridized sequentially
with the probes as shown. L32 is a loading control. (C) Plots
illustrate the level of gene expression in a single EB compared to
L32. Horizontal bars represent median values. *P<0.05
compared to untreated cells.
|
|
View larger version (12K):
[in a new window]
|
Fig. 3. VEGF increases the size and prolongs the life span of EryP. (A)
Size distribution of EryP. EBs were dissociated on day 7 and
assayed into hematopoiesis at 2x104 ml–1.
Colony size was determined by integrating the area of individual colonies
using the Northern Eclipse software. The range covered by columns are
equivalent and are determined arbitrarily. –VEGF, n=50; +VEGF,
n=53; P<0.003. (B) VEGF prolongs the life span of
EryP. Day-3 EryP, derived from either VEGF-treated (5 ng
ml–1) or untreated day-7 EBs, were transferred individually
into 96-well plates containing fresh medium. Viability was assessed by visual
inspection. Cells were considered nonviable when lysed or necrotic
(n=121). Histogram represents the percentage of day-3 EryP
that were viable 3 days after transfer. Histogram depicts pooled data from two
independent experiments. (C) VEGF stimulates blast-colony formation (BL-CFC).
Day-3 or day-3.5 EBs derived from R1 ES cells were assessed for BL-CFC in the
presence or absence of VEGF. Results are the mean±s.d. of
duplicates.
|
|
View larger version (42K):
[in a new window]
|
Fig. 4. VEGF activity determines the hematopoietic output during primitive
erythropoiesis. (A) Frequency of EryP per individual E8.5 (5-14
somite pairs) yolk sacs from Vegf+/+ (+/+),
Vegflo/+ (lo/+) and Vegflo/lo
(lo/lo) embryos. Yolk sacs were isolated, dissociated into
single-cell suspension and assayed for their content in hematopoietic
precursors. Plots illustrate pooled data from four individual litters and
n is the number of embryos of a corresponding genotype. Horizontal
bars represent median values. P<0.05 compared to either
heterozygous ( ) or wild-type embryos (*). (B)
Gene-expression analysis of E8.5 Vegflo hypomorph yolk
sacs. Globally amplified cDNA from single yolk sacs were probed for gene
expression. The stage of development of the embryos, expressed in somite pairs
(sp), is indicated at the top of each lane. No RT served as a control of
genomic DNA contamination. (C) Plots illustrate the level of gene expression
within individual yolk sacs as ratio of the indicated genes over L32
taken as an internal control. Horizontal bars represent median values.
|
|
View larger version (66K):
[in a new window]
|
Fig. 5. VEGF is essential for the survival of primitive erythrocytes. (A)
Single-cell suspensions of dissected E9.0-E9.5 yolk sacs were stained with
Annexin V-FITC (apoptosis) and TER119-PE (erythroid). Dead cells that stain
with 7-ADD were excluded from the analysis. (B) KI67 immunostaining of
E9.0-E9.5 Vegflo hypomorph yolk sacs. Homozygous
Vegflo/lo yolk sacs contain few blood islands, harboring
rare primitive erythroid cells (F,G) in contrast to heterozygote
Vegflo/+ (D,E) and wild-type Vegf+/+
(B,C) littermates. KI67-positive (brown precipitate), proliferating,
primitive, erythroid cells (arrows) are found at a slightly reduced frequency
in Vegflo/+ and Vegflo/lo yolk sacs.
n represents the number of primitive erythrocytes scored:
Vegf+/+, n=137; Vegflo/+,
n=237; Vegflo/lo, n=80. No staining was observed when
the primary antibody was omitted (data not shown). Nuclei were counterstained
with Methyl Green. Scale bar: 10 µm.
|
|
View larger version (45K):
[in a new window]
|
Fig. 6. Partial rescue of primitive erythropoiesis by the Scl transgene
(Scltg). (A) Analysis of Scl expression in
Sil-Scl transgenic E9.5 yolk sacs. Individual yolk sacs were analyzed
for endogenous and transgenic expression of Scl using quantitative
SYBR Green PCR. Histogram represents the average amount of Scl
mRNA±s.d., normalized according to the internal control (S16).
To estimate the relative levels of Scl expression from the transgene
and endogenous source, the molar amount of endogenous Scl in
wild-type mice was taken as 1. Scl+/+, n=4;
Scltg, n=6. (B) Apoptosis in TER119-positive cells.
TER119, Annexin V and 7-AAD staining were performed as in
Fig. 5. Data shown are from
three independent litters and are expressed as % of TER119-positive cells.
Note that the level of apoptotic death depends on the number of
Vegflo alleles and is attenuated by the Scl
transgene. *P<0.05; n is the number of embryos of a
corresponding genotype. (C) Analysis of erythroid genes in individual yolk
sacs was performed as described in Fig.
4. (D) Plots illustrate the level of gene expression within
individual yolk sacs as ratio of the indicated genes and the L32
internal control. Horizontal bars represent median values. Note that the
Scltg increases the level of Gata1 and ßH1
in homozygous Vegflo/lo yolk sacs.
P<0.01.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2004
