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HEMATOPOIESIS
Brief report
K-Ras is essential for normal fetal liver erythropoiesis
Waleed F. Khalaf, Hilary White, Mary Jo Wenning, Attilio Orazi, Reuben Kapur, and David A. Ingram
In vitro studies suggest that Ras activation is necessary for erythroid cell development. However, genetic inactivation of
the Ras isoforms H-Ras, N-Ras, and KRas in mice reportedly did not affect adult
or fetal erythropoiesis, though K-Rasⴚ/ⴚ
embryos were anemic. Given these discrepancies, we performed a more detailed analysis of fetal erythropoiesis in
K-Rasⴚ/ⴚ embryos. Day-13.5 K-Rasⴚ/ⴚ em-
bryos were pale with a marked reduction
of mature erythrocytes in their fetal livers.
The frequency and number of both early
(erythroid burst-forming unit [BFU-E]) and
late erythroid progenitors (erythroid
colony-forming unit [CFU-E]) were reduced in K-Rasⴚ/ⴚ fetal livers compared
with wild-type controls and displayed a
delay in terminal erythroid cell maturation. Further, K-Rasⴚ/ⴚ hematopoietic pro-
genitors had reduced proliferation in response to erythropoietin and Kit ligand
compared with control cells. Thus, these
studies identify K-Ras as a unique Ras
isoform that is essential for regulating
fetal erythropoiesis in vivo. (Blood. 2005;
105:3538-3541)
© 2005 by The American Society of Hematology
Introduction
Ras activation following binding of erythropoietin (EPO) and
kit-ligand (KitL) to their receptors is essential for the differentiation, proliferation, and survival of erythroid progenitors in vitro.1-10
In mammals there exist 3 homologous Ras proteins, H-Ras, N-Ras,
and K-Ras (reviewed in Scheele et al,11 Ehrhardt et al,12 and
Rebollo and Martinez13). While H-Ras⫺/⫺, N-Ras⫺/⫺, and doubly
mutant H-Ras⫺/⫺; N-Ras⫺/⫺ knockout mice have no overt hematopoietic cell phenotypes,14,15 K-Ras⫺/⫺ mice die between days 12.5
and 14 of gestation with fetal liver defects and evidence of
anemia.16,17 However, when a heterogenous population of K-Ras⫺/⫺
fetal liver cells was plated in methylcellulose assays, only a slight
decrease in erythroid progenitor cell frequency was observed.16
Based on these results, the anemia observed in K-Ras⫺/⫺ embryos
was attributed to a defect in the fetal liver microenvironment. Thus,
despite in vitro studies supporting a role for Ras in regulating
erythroid cell function, in vivo studies have not identified a Ras
isoform essential for erythropoiesis. Given these discrepancies, we
performed a more detailed analysis of erythropoiesis in K-Ras⫺/⫺
embryos by testing erythroid cell differentiation with a newly
developed flow cytometric assay18 and the erythroid colonyforming ability of a purified population of hematopoietic progenitor cells.
by the Indiana University Animal Care and Use Committee. The K-Ras
allele was genotyped by polymerase chain reaction as previously described.16 K-Ras⫹/⫺ mice were mated to produce day-13.5 K-Ras⫺/⫺ and
wild-type (WT) embryos. Fetal livers were isolated as previously described.19 Single-cell suspensions were prepared by pushing hepatic tissues
through a 23-gauge needle.
Fetal liver touch preparations
Fetal liver touch preparations were performed as previously described19
from day-13.5 WT and K-Ras⫺/⫺ embryos and stained with Wright Giemsa
(Dade Behring, Newark, DE). Photomicrographs of touch preparations
were taken with an Olympus DP11 microscope (Melville, NY).
Colony assays
Recombinant murine KitL and granulocyte macrophage-colony stimulating
factor (GM-CSF) were obtained from Peprotech (Rocky Hill, NJ), and EPO
was obtained from Amgen (Thousand Oaks, CA). Erythroid burst-forming
unit (BFU-E), erythroid colony-forming unit (CFU-E), and granulocytemacrophage colony-forming unit (CFU-GM) assays were performed exactly as previously described.19
C-kitⴙ cell isolation
Study design
Fetal liver cells were incubated with 5 ␮g of a fluorescein isothiocyanate
(FITC)–conjugated c-kit monoclonal antibody (Pharmingen, San Diego,
CA) per 106 cells, placed on ice for 30 minutes, pelleted, washed, and
resuspended in phosphate-buffered saline. C-kit⫹ cells were purified by
immunomagnetic bead enrichment as previously described.19
Mice and fetal hematopoietic cell isolation
Apoptosis, proliferation, and differentiation assays
K-Ras⫺/⫺ mice were obtained from Jackson Laboratories (Bar Harbor, ME)
in a 129/SV background. Studies were conducted with a protocol approved
Sorted c-kit⫹ fetal liver cells were stained with annexin V–FITC (Pharmingen) exactly per manufacturer’s protocol followed by flow cytometric
From the Department of Pediatrics, Herman B Wells Center for Pediatric
Research, Indiana University School of Medicine, Indianapolis, IN; and
Departments of Microbiology and Immunology, Hematology/Pathology, and
Biochemistry and Molecular Biology, Indiana University School of Medicine,
Indianapolis, IN.
O’Connor Award from the March of Dimes (5-FY02-254).
Submitted May 28, 2004; accepted December 26, 2004. Prepublished online
as Blood First Edition Paper, January 11, 2005; DOI 10.1182/blood-2004-052021.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by 1 KO8 CA096579-01 (D.A.I.). D.A.I. is a recipient of a Basil
© 2005 by The American Society of Hematology
3538
Reprints: David A. Ingram, Indiana University School of Medicine, Herman B
Wells Center for Pediatric Research, 1044 W Walnut St R4/470, Indianapolis,
IN 46202; e-mail: [email protected].
BLOOD, 1 MAY 2005 䡠 VOLUME 105, NUMBER 9
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BLOOD, 1 MAY 2005 䡠 VOLUME 105, NUMBER 9
DEFECTIVE ERYTHROPOIESIS IN K-Ras⫺/⫺ EMBRYOS
3539
analysis (FACS) as previously described.19 For survival assays, 10 000
c-kit⫹ fetal liver cells were plated in 96-well plates with no serum in the
presence of varying concentrations of either EPO or KitL alone or in
combination. After 48 hours in culture, cells were stained with annexin
V–FITC followed by FACS analysis. For thymidine incorporation assays,
10 000 c-kit⫹ fetal liver cells were plated in 96-well plates in serum-free
medium (X-VIVO 10; CAMBREX, Walkersville, MD) in the presence of
varying concentrations of either EPO or KitL alone or in combination. After
48 hours in culture, cells were pulsed with tritiated thymidine (New Life
Sciences, Boston, MA) for 16 to 24 hours, harvested on glass-fiber filters,
and ␤ emission was measured. In some experiments, freshly isolated c-kit⫹
cells were cultured in the presence of both KitL and EPO for 24, 48, and 72
hours to differentiate the cells to later stages of erythroid cell maturation.
After culture at each time point, survival and proliferation assays were
performed exactly as described in this section.
For the differentiation assay, fetal liver cells were stained with
anti-CD71–FITC (Pharmingen), anti–Ter-119–phycoerythrin (PE; Pharmingen), and anti–c-kit–allophycocyanin (APC; Pharmingen) antibodies followed by FACS analysis as previously established.18
Results and discussion
To determine the effect of K-Ras deficiency on fetal liver erythropoiesis, K-Ras⫹/⫺ mice were intercrossed and day-13.5 embryos
were harvested. As previously shown,16 K-Ras⫺/⫺ embryos and
fetal livers were small and pale compared with WT controls (data
not shown). K-Ras⫺/⫺ day-13.5 fetal liver touch preparations
demonstrated an increase in immature proerythroblasts and early
basophilic erythroblasts and a striking decrease in late basophilic,
chromatophilic, and orthochromatophilic erythroblasts (Figure
1A). Further, there were very few mature enucleated erythrocytes in K-Ras⫺/⫺ fetal livers (Figure 1A). In contrast, there was
a continuum of erythropoiesis in WT fetal livers with mature
enucleated erythrocytes identified throughout the fetal liver
(Figure 1A).
Based on this observation, we next compared erythroid cell
differentiation in vivo between K-Ras⫺/⫺ and WT fetal livers. We
used a recently developed flow cytometric assay, which readily
distinguishes various stages of erythroid cell differentiation by
analyzing the cell surface expression of CD71 and Ter-119.18
Consistent with our phenotypic observations (Figure 1A), we
observed a delay in terminal erythroid differentiation in K-Ras⫺/⫺
fetal livers compared with WT controls (Figure 1B). Specifically,
K-Ras⫺/⫺ fetal livers had a marked increase in early progenitor and
proerythroblasts (region 1 [R1]; 12% vs 6%) and in early basophilic erythroblasts (R2; 18% vs 6%) with a decrease in more
mature late basophilic erythroblasts (R3; 41% vs 65%) and
chromatophilic and orthochromatophilic erythroblasts (R4; 4% vs
12%; Figure 1B). To further characterize the delay in erythroid cell
maturation observed in K-Ras⫺/⫺ fetal livers, we costained fetal
liver cells with antibodies directed against CD71, Ter-119, and c-kit
and analyzed the cells by FACS. Interestingly, c-kit⫹ cells were
observed mostly in the R1 and R2 populations, which define early
progenitors, proerythroblasts, and early basophilic erythroblasts,
but there was no significant difference in the percentage of c-kit⫹
cells in the different gated populations (R1-R5) between the 2
genotypes (Figure 1C). However, since there is an increased
percentage of cells in R1 and R2 in K-Ras⫺/⫺ fetal livers (Figure
1B), these data indicate that there is an overall increase in the
percentage of c-kit⫹ cells in K-Ras⫺/⫺ fetal livers compared with
WT controls, which is consistent with an increased percentage of
early erythroid progenitors and a delay in erythroid maturation
Figure 1. Effect of K-Ras deficiency on fetal liver erythropoiesis. (A) Representative fetal liver touch preps from WT and K-Ras⫺/⫺ day-13.5 embryos. Touch
preparations were stained with Wright Giemsa. Numerous erythropoietic cells in all
stages of differentiation are observed in WT fetal livers and are represented as
follows: proerythroblasts by a blue arrow; basophilic erythroblasts by a black arrow;
polychromatophilic erythroblasts by a green arrow; orthochromatophilic erythroblasts
by a red arrow; and mature enucleated erythrocytes by a purple arrow. In contrast, a
marked increase in proerythroblasts (blue arrow) and basophilic erythroblasts (black
arrow) was observed in K-Ras⫺/⫺ fetal livers with a concomitant decrease in more
mature polychromatophilic and orthochromatophilic erythroblasts and a paucity of
enucleated erythrocytes. Slides were visualized under an Olympus BX51 microscope
(Olympus, Melville, NY) equipped with a UPlan FI 40 ⫻/0.75 objective lens.
Photomicrographs were acquired with an Olympus DP11 camera using MicroSuite
imaging software (Olympus). Five other experiments showed similar results. (B)
Representative flow cytometric analysis of erythroid differentiation in vivo. Freshly
isolated day-13.5 WT or K-Ras⫺/⫺ fetal livers were doubly stained with anti-CD71–
FITC and anti–Ter-119–PE. Regions R1 to R5 define distinct populations of erythroid
progenitor cells at different stages of differentiation as described previously.18
Primitive progenitor cells (including mature BFU-Es and CFU-Es) are shown in R1,
proerythroblasts and early basophilic erythroblasts in R2, early and late basophilic
erythroblasts in R3, chromatophilic and orthochromatophilic erythroblasts in R4, and
late orthochromatophilic and reticulocytes in R5, as previously described.18 Numbers
represent the percentage of cells within that region. Day-13.5 wild-type fetal livers
display normal distribution of erythroid cells at different stages of differentiation.
K-Ras⫺/⫺ fetal livers demonstrate a delay in differentiation at the basophilic erythroblast level: R2 to R3. Five other experiments showed similar results. (C) Percentage
of freshly isolated K-Ras⫺/⫺ or WT day-13.5 fetal liver cells expressing c-kit in regions
R1 to R5, which define distinct populations of erythroid progenitor cells at different
stages of differentiation. Freshly isolated day-13.5 WT or K-Ras⫺/⫺ fetal livers were
stained with anti-CD71–FITC, anti–Ter-119–PE, and anti–c-kit–APC antibodies as
described in “Apoptosis, proliferation, and differentiation assays.” The majority of
c-kit⫹ cells were identified in the more immature erythroid progenitor populations in
R1 and R2 and no significant difference in the percentage of c-kit⫹ cells was observed
in the different gated populations (R1-R5) between the 2 genotypes. Results
represent the mean percentage of c-kit⫹ cells and error bars represent the standard
error of the mean of 5 parallel independent experiments from embryos isolated from
the same litter.
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3540
KHALAF et al
observed in K-Ras⫺/⫺ day-13.5 embryos. Collectively, these results
argue that K-Ras is important for normal fetal liver erythroid cell
differentiation in vivo.
We next assayed for early (BFU-E) and late (CFU-E) erythroid
progenitors in WT and K-Ras⫺/⫺ fetal livers. The frequency of
BFU-Es and CFU-Es in day-13.5 K-Ras⫺/⫺ livers were reduced
compared with WT controls (Figure 2A). To directly test the effect
of K-Ras deficiency on erythroid colony formation independent of
the effects of the microenvironment, we performed BFU-E and
CFU-E assays with equal numbers of sorted WT and K-Ras⫺/⫺
c-kit⫹ cells. To confirm that there were no differences in cell
viability, an aliquot of sorted c-kit⫹ cells was stained with annexin
V to identify apoptotic cells. K-Ras⫺/⫺ and WT c-kit⫹ cells had a
similar percentage of annexin V–positive cells (data not shown). A
marked reduction in the frequency of both BFU-Es and CFU-Es
was observed in K-Ras⫺/⫺ c-kit⫹ cells compared with WT controls
(Figure 2B). Since the number of both BFU-Es and CFU-Es
generated from K-Ras⫺/⫺ c-kit⫹ cells were reduced, these data
argue that K-Ras activation is important for the proliferation and/or
survival of both early and late erythroid progenitors in response to
EPO and KitL. Interestingly, the number of myeloid colonies
(CFU-GMs) generated in response to GM-CSF from K-Ras⫺/⫺
c-kit⫹ fetal liver cells was reduced compared with WT controls
(30.17 ⫾ 2.29 vs 184.13 ⫾ 13.08; n ⫽ 5; P ⬍ .001), indicating
that K-Ras activation may also be essential for the development of
other hematopoietic cell lineages. Nevertheless, these results argue
that an intrinsic K-Ras⫺/⫺ hematopoietic progenitor cell defect
contributes at least in part to reduction of erythroid progenitors and
mature erythrocytes in day-13.5 K-Ras⫺/⫺ fetal livers.
Since these data establish that K-Ras is essential for erythropoiesis in vivo, we next tested whether the K-Ras⫺/⫺ erythroid
phenotype was also linked to either a decrease in proliferation
and/or survival of hematopoietic progenitors in response to KitL or
EPO. We performed thymidine incorporation assays using K-Ras⫺/⫺
and WT c-kit⫹ cells. Remarkably, K-Ras⫺/⫺ c-kit ⫹ cells displayed
a 50% to 60% decrease in proliferation in response to either EPO or
KitL alone and EPO and KitL in combination (Figure 2C). Similar
differences in proliferation between the 2 experimental genotypes
were obtained when WT and K-Ras⫺/⫺ c-kit⫹ cells were differentiated to later stages of erythroid cell maturation in vitro and
thymidine incorporation assays were performed with the different
populations of erythroid progenitors in response to either EPO or
KitL alone or in combination (data not shown). One explanation for
this result is that K-Ras⫺/⫺ c-kit⫹ cells were undergoing an
accelerated rate of apoptosis. To test this possibility, we compared
the survival of K-Ras⫺/⫺ and WT c-kit ⫹ cells in response to either
EPO and/or KitL in the absence of serum. Both K-Ras⫺/⫺ and WT
c-kit⫹ cells had a similar percentage of annexin V–positive cells at
the initiation of the experiment (data not shown). After 24, 48, and
72 hours in culture, no differences were detected in the percentage
of K-Ras⫺/⫺ or WT c-kit ⫹ apoptotic cells (annexin V–positive) in
response to either EPO or KitL alone or in combination (data not
shown). Thus, these data indicate that K-Ras activation is essential
for the proliferation of erythroid progenitor cells in response to
both EPO and KitL.
These studies establish a previously unrecognized role for K-Ras in
regulating fetal erythropoiesis in vivo. Specifically, K-Ras is essential for
the differentiation of erythroid progenitor cells to late basophilic
erythroblasts and the proliferation of hematopoietic progenitors in
response to EPO and KitL. Recent data demonstrate that Ras isoforms
undergo differential posttranslational modification (reviewed in Ehrhardt
et al,12 Bar-Sagi,20 and Hancock et al21) and compartmentalization
BLOOD, 1 MAY 2005 䡠 VOLUME 105, NUMBER 9
Figure 2. Erythroid colony formation and proliferation of K-Rasⴚ/ⴚ and WT
c-kitⴙ day-13.5 fetal liver cells in response to EPO and KitL. (A) Frequency of
erythroid progenitors in WT and K-Ras⫺/⫺ nonfractionated day-13.5 fetal liver cells.
Total nucleated fetal liver cells (40 000 cells/mL) were plated for growth of BFU-Es in
methylcellulose medium supplemented with 10 ng/mL KitL and 5 U/mL EPO. For
CFU-Es, total nucleated fetal liver cells (40 000 cells/mL) were plated in methylcellulose medium supplemented with 5 U/mL EPO. CFU-Es and BFU-Es were counted by
indirect microscopy at days 2 and 7 of culture, respectively. Results represent the
mean number of colonies and error bars represent the standard error of the mean of 5
parallel independent experiments, which were performed in triplicate from embryos
isolated from the same litter. *P ⬍ .05 by Student paired t test. (B) Frequency of
erythroid progenitors in c-kit⫹–sorted cells isolated from day-13.5 WT and K-Ras⫺/⫺
fetal livers. C-kit⫹ cells were isolated by immunomagnetic bead enrichment and
sorted as described in “C-kit⫹ cell isolation” to a purity of more than 85% as tested by
fluorescence cytometry (data not shown). C-kit⫹ cells (4000 cells/mL) were plated for
growth of BFU-Es in methylcellulose medium containing various concentrations of
KitL as indicated combined with a single concentration of EPO (5 U/mL). C-kit⫹ cells
(4000 cells/mL) were plated for growth of CFU-Es in methylcellulose medium
containing various concentrations of EPO alone as indicated. CFU-Es and BFU-Es
were counted by indirect microscopy at days 2 and 7 of culture, respectively. Results
represent the mean number of colonies per 4000 c-kit⫹ ⫾ SEM of 5 parallel
independent experiments. Colony assays were performed in triplicate from c-kit⫹
cells isolated from embryos isolated from the same litter. *P ⬍ .05 by Student paired t
test. (C) Proliferation of WT and K-Ras⫺/⫺ c-kit⫹ day-13.5 fetal liver cells in response to no growth factors, EPO and KitL alone, or in combination. Freshly isolated
c-kit⫹ fetal liver cells were plated in a 96-well plate at a concentration of 10 000
cells/well in serum-free medium with no additional growth factors or 10 ng/mL KitL
and 5 U/mL EPO in combination or alone. After 48 hours of culture, cells were pulsed
with tritiated thymidine and harvested 16 hours later for measurement of ␤ emission.
CPM indicates counts per minute. Results represent the mean thymidine incorporation ⫾ SEM of 5 independent experiments. Thymidine incorporation assays were
performed in triplicate from c-kit⫹ cells isolated from embryos harvested from the
same litter. *P ⬍ .05 by Student paired t test.
within the cell (reviewed in Prior and Hancock,22 Hancock,23 and Chiu
et al24) and that individual Ras isoforms can differentially activate
discrete signaling pathways (reviewed in Ehrhardt et al12). Our data
provide evidence that single Ras isoforms may also provide unique
signals in hematopoietic cells in vivo.
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BLOOD, 1 MAY 2005 䡠 VOLUME 105, NUMBER 9
DEFECTIVE ERYTHROPOIESIS IN K-Ras⫺/⫺ EMBRYOS
3541
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2005 105: 3538-3541
doi:10.1182/blood-2004-05-2021 originally published online
January 11, 2005
K-Ras is essential for normal fetal liver erythropoiesis
Waleed F. Khalaf, Hilary White, Mary Jo Wenning, Attilio Orazi, Reuben Kapur and David A. Ingram
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