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First published online 11 June 2008
doi: 10.1242/dev.020131
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1 Pacific Vascular Research Laboratory, Division of Vascular Surgery,
Departments of Surgery and Anatomy, University of California, San Francisco,
CA 94143, USA.
2 Genetics and Stem Cell Laboratory, Swiss Institute for Experimental Cancer
Research, Ch. des Boveresses 155, CH-1066, Epalinges, Switzerland and Ecole
Polytechnique Fédérale de Lausanne (EPFL), School of Life
Sciences, CH-1015 Lausanne, Switzerland.
** Author for correspondence (e-mail: rong.wang{at}ucsfmedctr.org)
Accepted 16 May 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: c-myc, Angiogenesis, Vasculogenesis, Mouse, Hematopoiesis, Myc, Vascular development
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Evan et al., 2005;
Grandori et al., 2000;
Pelengaris et al., 2002), and
its overexpression is highly tumorigenic in many types of tissues in animals.
A large body of work performed in cultured cells shows that c-Myc is expressed
broadly and functions as a central regulator of normal cellular programs,
including cell proliferation, differentiation, growth, survival and migration
in many cell types (Grandori et al.,
2000). Although these studies have made tremendous contributions
to our understanding of the cellular mechanisms underlying the oncogenic
effects of c-Myc, the physiological function of this protein remains largely
unknown.
c-myc-null embryos exhibit severe developmental abnormalities in a
wide range of organs and die early in gestation before 10.5 days post coitum
(dpc) (Davis et al., 1993;
Trumpp et al., 2001),
supporting the notion that c-myc is essential for a broad range of
organ development. Recent studies suggest that c-Myc is required for the
proliferation of progenitor cells and the self-renewal of stem cells
(Murphy et al., 2005). In the
intestine, c-Myc is expressed in the proliferative zone of intestinal crypts,
where putative intestinal stem cells reside, and is essential for the
formation of these crypts (Bettess et al.,
2005; Muncan et al.,
2006). Similarly, in the skin epidermis, c-Myc is expressed in the
proliferative basal layer and bulge region, where stem and progenitor cells
are located (Bull et al.,
2001). These new in vivo findings suggest that c-myc is
uniquely required in the stem and progenitor cell compartments.
The role of c-myc in the development of the vascular system is of
particular interest because it is crucial not only for all aspects of normal
tissue function but also for pathological tumor growth and survival.
Endothelial cells (ECs) line blood vessels and are the primary cell type
responsible for blood vessel function and regeneration. Hematopoietic cells
(HCs) give rise to the blood cells of the circulatory system. Differentiation
of these two lineages first occurs in yolk sac blood islands, where ECs and
HCs may arise from a common mesoderm-derived precursor: the hemangioblast
(Cumano and Godin, 2007;
Eichmann et al., 2002;
Ema and Rossant, 2003). During
vascular morphogenesis, ECs coalesce to assemble a primitive vascular network
composed of a capillary plexus with uniform caliber and honeycomb appearance.
This formation of blood vessels by de novo EC differentiation is known as
vasculogenesis (Adams and Alitalo,
2007; Carmeliet,
2005). The primitive capillary plexus subsequently undergoes
growth and remodeling to shape the mature vascular tree. Angiogenesis is the
process of new blood vessel growth from existing vessels
(Folkman, 2006;
Hanahan and Folkman, 1996;
Thurston et al., 2007). c-Myc
has been shown to regulate angiogenesis by promoting the expression of
pro-angiogenic factors such as VEGF in stromal cells, while inhibiting the
expression of the anti-angiogenic factor thrombospondin 1
(Baudino et al., 2002;
Dews et al., 2006;
Knies-Bamforth et al., 2004;
Mezquita et al., 2005;
Shchors et al., 2006;
Watnick et al., 2003). c-Myc
is reportedly required for vasculogenesis during development, as
c-myc-null embryos have no detectable blood vessels
(Baudino et al., 2002).
However, whether c-Myc in ECs plays an essential role in vasculogenesis or
angiogenesis is currently unknown.
To ascertain the cell type-specific role of c-Myc during vascular development, we generated conditional knockouts (CKs) of the c-myc gene in c-mycflox/flox mice using cell lineage-specific Cre lines. We were surprised to find that c-myc was not required for vasculogenesis, and that deleting c-myc in a majority of ECs was compatible with early embryo survival. By contrast, c-myc deletion was detrimental to hematopoietic lineages during development, and c-myc deletion in these lineages was sufficient to cause vascular developmental defects.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; de Boer et al.,
2003; Gustafsson et al.,
2001; Hayashi et al.,
2002; Iljin et al.,
2002; Trumpp et al.,
2001). All animals were treated in accordance with the guidelines
of the University of California San Francisco (UCSF) Institutional Animal Care
and Use Committee.
Materials
Details of antibodies and real-time PCR primers used can be provided on
request.
Imaging of embryos, whole-mount immunofluorescence staining, and EC isolation and culture
Previously established procedures
(Braren et al., 2006) were
followed.
Whole-mount lacZ staining
lacZ staining of embryos and yolk sacs was as previously described
(Carpenter et al., 2005).
Samples were fixed in 4% paraformaldehyde (PFA) overnight after lacZ
staining. Specimens were then embedded in paraffin and sectioned at 5 µm.
Sections were stained with Eosin and visualized using a Zeiss Axioskop 2 Plus
microscope (Zeiss, Thornwood, NY). Images were captured using a DC 300 camera
and IM50 software (Leica, San Jose, CA).
Cell proliferation and TUNEL assays
Cell proliferation and apoptosis were evaluated as previously described
(Braren et al., 2006).
TUNEL+CD31+ ECs and TUNEL+CD31-
cells were counted, and the ratio of TUNEL+CD31+ cells
to total CD31+ ECs was obtained. Four pairs of embryos from two
different litters were examined. Statistical analysis was performed using the
t-test.
EC motility assay
ECs isolated from 10.5 dpc embryos were plated on a six-well plastic plate
coated with 10 µg/ml fibronectin and cultured overnight, then labeled for 3
hours with Dil
(1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine
perchlorate)-labeled acetylated low-density lipoprotein (DiI-Ac-LDL,
Biomedical Technologies, Stoughton, MA) at 2.5 µg/ml in medium. Time-lapse
microscopy was performed using a Marianas time-lapse imaging system
(Intelligent Imaging Innovations, Santa Monica, CA). EC migration was recorded
for 4 hours at 6-minute intervals. xy coordinates of individual ECs
were tracked with Slidebook software (Intelligent Imaging Innovations, Santa
Monica, CA).
Fetal liver cell-isolation and flow cytometry analysis
Fetal livers at 11.5 dpc were dissociated mechanically and passed through a
40 µm nylon mesh. Cells were collected in 10% FBS/0.5% BSA/1x
PBS-calcium and magnesium free (CMF). Cell viability was determined using a
Trypan Blue dye exclusion assay. For flow cytometry analysis, cells
(3-5x105) were diluted into 100 µl 5% FBS/0.5%
BSA/1x PBS-CMF, and the antibody-cell suspension was incubated on ice
for 30 minutes. A BD LSRII FACS Machine (BD Biosciences, San Diego, CA) was
used to perform flow-cytometry, and FlowJo software was used for data
analysis. Propidium iodide (1 µg/ml) staining was used to exclude dead
cells.
Collection of embryonic peripheral blood cells
Embryonic peripheral blood (PB) was isolated by opening the embryonic
vitelline vessels, dorsal aortae and the heart to release blood cells
completely. The cells were passed through a 40 µm nylon mesh before
use.
Immunofluorescence staining of sections and isolated cells
Rehydrated paraffin sections were blocked with 5% donkey serum in PBS for 2
hours at room temperature. They were then incubated with primary antibody at
4°C overnight, washed three times with PBS, followed by 1 hour secondary
antibody incubation at 4°C in blocking solution (2% BSA, 3% normal donkey
serum, 0.01% Triton X100, 1xPBS). Samples were washed with PBS and
mounted with Vectashield containing DAPI (4', 6
diamidino-2-phenylindole; Vector Laboratories. Burlingame, CA). Cells grown on
six-well plates were fixed in 4% PFA/PBS for 20 minutes and permeabilized in
0.1% Triton X-100/1xPBS/2%BSA for 10 minutes at room temperature before
blocking (2% BSA/0.01% of Triton X100/1xPBS). Images were captured using
either air lenses or a 63x Anchroplan water immersion lens, and a Zeiss
Axiovert2 Plus microscope equipped with a Sensicam CCD camera and Slidebook
software (Intelligent Imaging Innovations, Santa Monica, CA).
Allantoic explants
Allantoic explant was performed as described
(Braren et al., 2006).
Allantoises were isolated at E8.0 at the 6- to 8-somite stage and cultured for
24-48 hours on FN-coated dishes. Cultures were stained with anti-CD31 as
described above.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Trumpp et al., 2001).
Multi-organ failure, including circulatory defects, is believed to underlie
this early developmental arrest, although the specific cell lineages affected
are largely unknown. We therefore analyzed c-myc-/-
embryos specifically for vascular defects, using whole-mount immunostaining
against CD31, an EC-specific marker. As previously reported, these mutant
embryos were developmentally retarded and displayed major developmental
defects. However, we were surprised to find that major vessels such as the
dorsal aorta had developed by 8.75 dpc. Smaller inter-somitic and cranial
vessels had also formed in the embryo proper, although they were
underdeveloped compared with wild-type vessels
(Fig. 1A,B). In the yolk sac,
primitive vascular networks had formed, although the mutant yolk sac contained
more vascular plexuses and less organized hierarchal branches than the control
(Fig. 1C,D). At 10.25 dpc,
although the control vasculature had matured to an elaborate and
well-organized system (Fig.
1E,G), the c-myc-/- embryos retained a
primitive vasculature in the head (Fig.
1F) and the yolk sac (Fig.
1H). Nonetheless, embryo cross-sections revealed that, like
controls, c-myc-/-embryos had ECs lining their dorsal
aortae, common cardinal veins and other blood vessels
(Fig. 1I,J). These results
indicate that, in contrast to a previous report
(Baudino et al., 2002),
c-myc-/- embryos contained differentiated ECs that were
capable of assembling a primitive vasculature. Subsequent vascular remodeling,
however, was defective in the mutants.
Because the detection of ECs in the c-myc-/- mutant was
surprising, we examined the vasculature in another mutant embryo, in which
c-myc is deleted in the entire embryo proper but retained in the
placenta. We created a CK using the Sox2-Cre mouse line, in which Cre
is active in all cells of epiblast origin, including the entire embryo proper,
the yolk sac mesoderm, the amnion membrane and the embryonic vessels in the
placenta, but not the visceral endoderm or the extra-embryonic ectoderm
(Vincent and Robertson, 2003).
We verified Cre expression throughout the entire embryo by Cre reporter assay
(data not shown). The Sox2-Cre;c-mycflox/- mutant progeny
displayed growth retardation by 11.5 dpc and died between 11.5 and 12.5 dpc.
These embryos, unlike the null mutants, appeared grossly normal without major
organ defects before E10.5 (data not shown). However, at E11.5 they were
anemic (Fig. 1L,N) and
displayed abnormal vasculature (Fig.
1P,R,T). Anti-CD31 staining revealed abundant ECs in mutant yolk
sacs and embryos proper (Fig.
1O-T), demonstrating that the mutant embryos were not defective in
EC differentiation, although capillary remodeling was defective. Mutant yolk
sac microvessels were more primitive, comprising disorganized capillary
plexuses with larger intercapillary spaces than in the controls. Major
vitelline vessels were narrower and underdeveloped in the mutant
(Fig. 1O,P). Similarly, head
capillaries were more primitive. The carotid arteries were less elaborate and
the primitive head veins were narrower compared with controls
(Fig. 1Q-T). These findings
confirmed that c-myc is not required for the onset of vasculogenesis
in embryos. However, it is essential for subsequent vascular
morphogenesis.
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), and
c-Myc promotes trophoblast proliferation
(Erlebacher et al., 2004). We
found that by 8.25 dpc, c-myc-/- placentas contained fewer
trophoblasts, exhibiting defective placental morphology and cellular
composition lacking trophoblast integrity. In addition, the mutant chorionic
plate was thinner, and the ectoplacental cone was not integrated (see Fig. S1
in the supplementary material). These findings suggest that c-myc is
required for placental development, and that the cellular function of
c-myc in this organ requires future investigation. In this report, we
have focused on the function of c-myc in the embryo proper.
Loss of c-myc in endothelial and hematopoietic cells induces similar vascular defects as loss of c-myc in the entire embryo proper
To determine whether eliminating c-myc specifically from the
circulatory system would result in developmental and vascular defects, we
crossed c-mycflox/flox mice with
Tie2-Cre;c-myc+/- mice, in which Cre is active in EC and
HC lineages starting as early as 7.5 dpc in the common progenitor of these two
lineages (Braren et al., 2006).
To assess the efficiency of Tie2-Cre-mediated c-myc
deletion, we measured nuclear-specific c-Myc expression in isolated ECs by
immunostaining (see Fig. S3 in the supplementary material). Quantitative
analysis showed that virtually no (0.62%) mutant ECs and 91.2% of control ECs
had c-Myc staining by 10.5 dpc (Fig.
2M). These experiments demonstrate the success of c-myc
deletion.
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Anti-CD31 staining on 11.5 dpc Tie2-Cre CK embryos also revealed a very similar vascular phenotype to the Sox2-Cre CK, with abundant ECs but more primitive capillaries and smaller major vessels in mutant yolk sacs and heads compared with the controls (Fig. 2G-L). Quantitative analysis of the yolk sac vascular defects is summarized in Fig. S2 (see supplementary material). These findings from Tie2-Cre CK embryos confirm that vasculogenesis occurred without c-myc, but further capillary remodeling was defective. Additionally, the allantois is vascularized by vasculogenesis, and we did not detect any apparent vascular defects in the Tie2-Cre CK allantoic explants (Fig. 3A,B), further suggesting that vasculogenesis occurs in the absence of c-myc.
Expression of N-Myc (Mycn - Mouse Genome Informatics), another Myc family
member, driven by the c-myc promoter can functionally replace c-Myc
activity in vivo (Malynn et al.,
2000). To rule out the possibility that n-myc expression
might compensate for the loss of c-myc in ECs, we stained purified
ECs from 10.5 dpc Tie2-Cre CKs with anti-N-Myc antibodies (Fig. S3 in
the supplementary material). We found that N-Myc was expressed in a small
fraction (7.1-8.4%) of ECs, but that this fraction was independent of the EC
genotype (Fig. 2N). To rule out
a possible compensatory effect from L-myc, we analyzed L-myc
expression by real time RT-PCR in Sox2-Cre CK yolk sacs. Whereas
c-myc expression was significantly reduced, L-myc expression was unchanged in
the mutant (data not shown). These results demonstrate that neither N-Myc nor
L-Myc is likely to compensate functionally for c-Myc in ECs.
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To characterize defects in the HC lineage, we counted the peripheral red
blood cells (Ter119+), hematopoietic cells (CD45+) and
myeloid cells (CD11b+) from yolk sacs and embryos and found that by
10.5 dpc Tie2-Cre; c-mycflox/- mutants exhibited
a 38-fold reduction in total blood cell number, a 13-fold reduction in
CD45+ cells and a fourfold reduction in CD11b+ cells
(Fig. 2O). Hematoxylin and
Eosin staining on paraffin sections also confirmed a nearly complete absence
of blood cells by 11.5 dpc (data not shown). Hemogenic ECs that reside in the
ventral side of the dorsal aorta at around 10.5 dpc are thought to give rise
to HSCs (de Bruijn et al.,
2002; Taoudi and Medvinsky,
2007). We examined serial cross-sections of dorsal aortae from
four pairs of embryos at 10.5 dpc. Cells located at the ventral wall of the
aorta and morphologically resembling hemogenic ECs were seen in all controls
but in none of the mutants (Fig.
3E,F). These findings suggest that removing c-myc from
ECs and HCs is sufficient to induce the hematopoietic, angiogenic and survival
defects observed in embryos harboring a global c-myc deletion.
c-myc deletion in HCs is sufficient to induce vascular defects and embryonic lethality
To delineate the effect of c-myc deletion in hematopoietic
lineages on vascular development, we examined embryos in which c-myc
had been deleted specifically in HC lineages using Vav-iCre. Vav-iCre
has been shown to mediate gene excision in adult HCs
(de Boer et al., 2003). We
thus analyzed Vav-iCre activity in embryos using a
Rosa26R-lacZ reporter according to our established method
(Braren et al., 2006). At 11.5
dpc, we found that Vav-iCre was active almost exclusively in fetal
liver HCs (Fig. S4A,C in the supplementary material) and in some circulating
blood cells in the yolk sac (Fig. S4B in the supplementary material), but not
in the endothelium or any other tissues (see Fig. S4D in the supplementary
material). This result indicates that Vav-iCre mediated
c-myc deletion in HCs without affecting ECs or any other cell types.
We assessed Vav-iCre activity in individual embryos by FACS analysis
in 11.5 dpc fetal liver HCs using the Rosa26YFP reporter
(Srinivas et al., 2001). The
fraction of HCs expressing Cre varied among individual embryos in all three HC
lineages tested, with averages around 50% (see Fig. S4K in the supplementary
material).
About 60% of Vav-iCre;c-myc flox/- mutants (27/43) appeared anemic at 11.5 dpc (Fig. 4B), with paler and smaller fetal livers compared with controls (see Fig. S4I,J in the supplementary material). A similar % of mutants died around 12.5dpc, a stage similar to the Tie2-Cre and Sox2-Cre CKs. About 16% of mutants (7/43) showed various degrees of hemorrhaging in the embryo proper (see Fig. S4E,G in the supplementary material). About 21% of mutants (9/43) survived through 12.5 dpc but developed anemia and died by 15.5 dpc. None of the Vav-iCre mutants survived to birth. It is likely that non-uniform Cre activity among individuals resulted in these variable phenotypes (see Fig. S4K in the supplementary material). Nonetheless, deletion of c-myc solely in HCs resulted in complete embryonic lethality of all embryos examined.
To visualize the vasculature of the Vav-iCre;c-myc flox/- mutant embryos, we performed whole-mount anti-CD31 staining on 11.5 dpc embryos. We found that the vascular defects in these anemic embryos resembled those in Tie2-Cre- and Sox2-Cre CKs (see Fig. 1P,R,T, Fig. 2H,J,L). Although the overall vascular patterning in Vav-iCre;c-myc flox/- embryos proper and yolk sacs was in place, the vessels were narrower and the vascular network appeared underdeveloped and primitive compared with controls (Fig. 4D,F,H). Taken together, these results suggest that c-myc deletion in HC lineages alone likely accounts for the anemia, embryonic lethality and vascular developmental defects induced by c-myc deletion in ECs and HCs combined.
To quantify the hematopoietic defects in 11.5 dpc Vav-iCre CKs, we
performed HC counts in peripheral blood
(Fig. 4I) and fetal liver cells
(Table 1). We found a several
fold decrease of cells (7.6 fold in Ter119+ cells, 4.3 fold in
CD45+ and 4.6 fold in CD11b+ cells) in the mutant
peripheral blood. The mutants' fetal livers had cytopenia and contained
12.5-fold fewer cells than their littermate controls. In addition, we used Lin
markers, containing CD3e, CD11b, CD45R/B220, Ter119, Ly-6G and Ly-6C to label
the committed hematopoietic lineages, which include T lymphocytes, B
lymphocytes, monocytes/macrophages, NK cells, erythrocytes and granulocytes.
We found the proportion of committed (Lin+) cells was reduced while
that of uncommitted cells (Lin-) was increased in
Vav-iCre;c-mycflox/- embryos. Among the
Lin- cells, the proportion of KLS-HSCs (Kit+,
Lin-/lo, Sca1+)
(Ivanova et al., 2002) in the
Vav-iCre;c-mycflox/- fetal liver was increased,
while the proportion of Kithi, Lin- cells was
significantly decreased. In summary, these results demonstrate that
elimination of c-myc in HCs by Vav-iCre compromised
definitive hematopoiesis.
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) (see
Fig. S5 in the supplementary material). We verified Tie1-Cre activity
using the Rosa26R reporter and double immunostaining for CD31 and
β-galactosidase. Approximately 80% of CD31-positive cells were Cre active
at 10.5 dpc (see Fig. S5 in the supplementary material). We also characterized
Tie1-Cre expression in HCs by FACS using the RosaYFP
reporter. Cre was active in 
54% of myeloid cells, 26% of erythroid cells
and 15% of lymphocyte precursor cells at 11.5 dpc (Fig. S6 in the
supplementary material). These results suggest that Tie1-Cre mediated
c-myc deletion in the majority of ECs and a subset of HCs. By 12.5 dpc, we did not detect any defects in Tie1-Cre;c-mycflox/- mutants. By 17.5 dpc we observed anemic, dying mutant embryos. Remarkably, half of the mutants survived to birth and a third to post-weaning (Table 2). Surviving mutant adults appeared normal. These results demonstrate that c-myc deletion in a majority of ECs is compatible with embryo survival.
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To determine whether loss of c-myc affected EC survival in vivo, we performed TUNEL assays on cryosections of yolk sacs. We found a significant increase in TUNEL staining in Tie2-Cre CKs compared with the controls at 10.5 dpc (Fig. 6B). However, increased TUNEL staining affected both ECs and non-ECs, even though non-ECs had a functional c-myc gene. These data suggest that the increase in EC death could be a secondary effect resulting from general poor embryo health rather than a direct result of c-myc deletion in these cells. In summary, we were unable to detect significant changes in cell proliferation and survival that could be ascribed to loss of c-myc specifically in ECs.
To examine the morphology and behavior of c-myc-deficient ECs, we isolated ECs from Tie2-Cre CKs at 10.5 dpc and cultured them for 5 hours on fibronectin-coated culture plates. Using anti-CD31 and phalloidin staining, we observed neither morphological differences nor changes in the organization of the actin cytoskeleton between control and c-myc-null ECs (Fig. 6C,D).
Normal angiogenesis relies on the ability of ECs to migrate
(Beck and D'Amore, 1997). To
assess the motility of c-myc-deficient ECs, we performed time-lapse
video-microscopy of c-myc-null and control ECs isolated from 10.5 dpc
embryos and cultured on fibronectin. The paths of both mutant and control ECs
were random and indistinguishable from one another, as shown by our
measurements of net path length and average speed
(Fig. 6E,F,G). These results
demonstrate that c-myc-null ECs are not defective in cell motility in
vitro.
To further address the angiogenic potential of c-myc-null ECs, we tested their ability to form endothelial tubes in vitro (Fig. 6H,I). Because this assay requires a large number of ECs, we isolated ECs from the vena cava of adult c-mycflox/flox mice and removed their c-myc gene using an adenovirus-mediated Cre (AdCreGFP) deletion system. FACS analysis of GFP expression in the cultured ECs showed that the efficiency of adenovirus infection was 98% (Fig. 6J), and PCR analysis of genomic DNA confirmed that the c-myc gene was excised in the majority of the cells (Fig. 6K). However, we found no statistically significant difference in the number of branch points (data not shown) or the lengths of tubes formed by mutant and control ECs on the Matrigel surface (Fig. 6I,J). These findings suggest that c-myc-null ECs are not defective in cell migration or capillary morphogenesis.
Deletion of c-myc in HCs leads to reduction of proangiogenic factors crucial for vascular morphogenesis
HCs have been increasingly recognized as significantly contributing to
angiogenesis by modulating the production of proangiogenic factors
(Tordjman et al., 2001;
Kopp et al., 2006;
Shojaei et al., 2007). We
therefore examined the mRNA levels of various proangiogenic factors, including
Pdgfa, Mmp2 and Il1b,in whole embryos, using
quantitative-PCR analysis. We found that mRNA levels of these genes were
significantly decreased in the anemic
Vav-iCre;c-mycflox/- embryos compared with their
control littermates (Fig. 7A).
By contrast, levels of VEGF transcripts were dramatically increased,
indicating a hypoxic response in the embryos suffering anemia (see
Fig. 4A,B). Similarly, elevated
VEGF protein was found in Tie2-Cre;c-mycflox/-
embryos (Fig. 7B). These
results demonstrate that c-Myc deficiency-induced hematopoietic defects may
have reduced expression of certain proangiogenic factors, thereby hindering
normal vascular morphogenesis.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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c-Myc plays an essential role in embryonic hematopoiesis
Fetal hematopoiesis begins with primitive hematopoietic differentiation in
blood islands of the yolk sac at 7.5 dpc and lasts until 10.5 dpc in mice.
Definitive hematopoiesis, which generates enucleated erythrocytes among other
hematopoietic lineages like HSCs, starts at 10.5 dpc in the
aorta-gonad-mesonephros region. Concurrently, HSCs colonize the developing
fetal liver. The murine placenta also harbors HSCs during midgestation. Around
birth, hematopoiesis translocates to the bone marrow (BM). Adult and fetal
hematopoiesis differ in the types of niches in which they occur, as well as in
the capacity of adult versus fetal HSCs to renew, proliferate and
differentiate (Cumano and Godin,
2007; Mikkola and Orkin,
2006; Wilson and Trumpp,
2006). The role of c-Myc in adult hematopoiesis in BM has been
reported (Wilson et al.,
2004). Our experiments demonstrate the essential function of c-Myc
in fetal hematopoiesis.
Our data suggest that HCs undergo primitive differentiation in the absence
of c-myc. At 9.5 dpc, prior to definitive hematopoiesis, the majority
of Tie2-Cre CKs were indistinguishable from the controls, with
blood-filled circulatory systems. This phenotype is in sharp contrast to that
of embryos lacking Scl, a gene required for the differentiation of
primitive hematopoietic cells. Scl-null embryos are devoid of blood
cells with no sign of hematopoiesis at 9.5 dpc
(Robb et al., 1995). One
concern is whether Tie2-Cre mediated c-myc deletion occurs
early enough to assess its requirement in primitive hematopoiesis. We have
previously reported that this Tie2-Cre is active early in the blood
island precursors and mediates efficient gene excision by 9.5 dpc
(Braren et al., 2006). The
presence of blood cells in both the c-myc null
(Davis et al., 1993) and
Sox2-Cre CKs (data not shown) further supports the idea that
primitive hematopoiesis occurs in the absence of c-myc. From 9.5 dpc,
the Tie2-Cre CKs developed progressive cytopenia. The likely cause of
the primitive hematopoietic failure is the reduced survival of HCs
(Dubois et al., 2008). Thus,
c-myc is not required for the initiation of primitive hematopoiesis
but is essential to sustain primitive hematopoiesis.
|
). Furthermore, we show that Vav-iCre mediates gene
excision specifically in fetal liver HCs. The fact that the Vav-iCre
CK developed severe cytopenia demonstrates that c-myc in HCs is
essential for definitive hematopoiesis.
The absence of c-Myc in definitive hematopoiesis seems to affect the
differentiation of HSCs, resembling c-myc-deficient adult
hematopoiesis in BM (Wilson et al.,
2004). The proportion of uncommitted HCs, KLS-HSCs and
Kitlow progenitor cells increased in the Vav-iCre fetal
liver. However, the proportion of Kithi progenitor cells decreased.
This reduction could be a genuine decrease of Kithi progenitors in
mutants or a loss of cell surface Kit expression on otherwise functional HSCs,
as has been observed following myeloid ablation
(Randall and Weissman, 1997).
However, the proportion of the committed HCs, including Ter119+,
CD45+ and CD11b+ lineages, decreased significantly in
Vav-iCre CKs. In addition, the total number of isolated fetal liver
cells, which are composed primarily of HCs, was decreased. These data suggest
that the mutant HSCs and progenitor cells can survive and divide but
subsequent differentiation into HCs are defective. Our findings provide
crucial evidence that c-Myc is required in a cell-autonomous fashion for HSC
differentiation. This finding is complementary to the finding in
Sox2-Cre CKs (Dubois et al.,
2008).
The fact that mutant embryos in which c-myc is deleted in the
entire embryo proper but not the placenta survived 2 additional days beyond
the survival of the complete null embryo shows that c-myc is
essential for placental development. It is currently unknown which specific
cell lineage(s) c-Myc may function in and what precise role c-Myc may play in
the placenta. However, this finding is intriguing in light of recent
discoveries that the placenta is an active site for HSC development
(Gekas et al., 2005;
Mikkola et al., 2005). Given
the crucial function of c-Myc in both fetal and adult hematopoiesis, it is
plausible that c-Myc may function in placental HSCs. The c-Myc CKs described
here may serve as an excellent model to elucidate the molecular control of
placental hematopoiesis.
c-Myc is not required for embryonic vasculogenesis
Previous reports suggest that c-myc is required for vasculogenesis
(Baudino et al., 2002) and
downregulation of c-myc in cultured ECs leads to cellular senescence
(Guney and Sedivy, 2006). By
contrast, we show here that vasculogenesis occurs in the absence of
c-myc. Our method of gene excision is efficient, leading to the
deletion of the entire coding region for c-Myc
(Trumpp et al., 2001). We
therefore respectfully disagree with this earlier conclusion. Gene disruption
in the two studies was achieved by targeting a similar region of
c-myc, so the reason for the discrepancy between our observations and
those of Baudino et al. are currently unclear. However, we confirmed our
results in three independent c-myc-deficient mutants (c-myc
null, Sox2-Cre and Tie2-Cre CKs), and the presence of ECs in
c-myc deleted embryos was also verified in a different laboratory
(Dubois et al., 2008). We
conclude that c-myc is not required for vasculogenesis in
embryos.
At the cellular level, c-myc-deficient ECs did not exhibit
detectable defects in cell proliferation, survival, migration or even
capillary morphogenesis. These results are in contrast to the report that
c-Myc is essential for EC proliferation in culture
(Guney and Sedivy, 2006). We
performed proliferation assays using primary ECs to closely mimic in vivo
conditions. In addition, neither N- nor L-Myc compensated for the loss of
c-Myc. We also show that about one third of Tie1-Cre CKs, in which
c-Myc was deleted in the majority of ECs, survived into adulthood without
apparent abnormalities. This result suggests that widespread deletion of
c-myc in the endothelium is compatible with survival. Taken together,
our observations suggest that abrogating c-Myc in ECs may not disrupt
angiogenesis, and c-Myc likely regulates angiogenesis through a
non-cell-autonomous fashion.
Hematopoietic abnormalities caused by c-myc deletion lead to defects in angiogenesis
Although a primitive vascular network formed in the absence of
c-myc, its angiogenic remodeling into a complex vascular tree was
abnormal. Because c-Myc-deficient ECs appear to function normally, we propose
that defective HCs cause the vascular defects observed in our c-myc
CK embryos. Supporting this notion, vascular defects in the Vav-iCre
CKs, where c-myc is deleted specifically in the hematopoietic
lineage, resembled those in the Tie2-Cre CK embryos.
HCs are known to affect angiogenesis through hemodynamic influence
(Lucitti et al., 2007) and
oxidative stress, such as hypoxia (Jones
et al., 2004; Ramirez-Bergeron
et al., 2006). The reduced hematocrit in the mutants likely
changes the viscosity of the blood, and hence alters the hemodynamic forces
required for growth and maintenance of vessel size
(Lucitti et al., 2007).
Moreover, both Vav-iCre and Tie2-Cre CK embryos were anemic
by 11.5 dpc. Hypoxia was evident by elevated VEGF levels, a common consequence
of embryos in hypoxic conditions. Hypoxia causes pan-tissue damage via
apoptosis (Graven et al.,
1993). Therefore, both low hematocrit and the hypoxia-mediated
apoptosis could contribute to the vascular defects observed in the
c-myc mutants.
However, lack of hemodynamic force and increased hypoxic stress are not the
only explanation for the absence of vascular remodeling in c-myc
mutants. HCs secrete factors capable of promoting angiogenesis in a paracrine
manner (Okamoto et al., 2005;
Okuda et al., 1996). Lack of
proangiogenic factors from HCs are responsible for angiogenic defects in
Arnt (Ramirez-Bergeron et al.,
2006) and Aml1
(Takakura et al., 2000)
mutants. As Tie-2Cre;c-mycflox/- embryos have
hematopoietic defects, they might also lack HC-derived proangiogenic factors,
which could explain their angiogenesis defects.
Consistent with this hypothesis, pale
Vav-iCre;c-mycflox/- embryos exhibited a significant
decrease of Il1b and Mmp2 mRNA at 11.5 dpc. IL1β is
secreted primarily from monocytes and macrophages. In the anemic
Vav-iCre;c-mycflox/- embryos, the significant decrease of
CD11b+ myeloid cells in fetal livers and peripheral blood likely
explains the decrease of IL1β expression and secretion. IL1β and
MMPs were recently found to form an axis to regulate the bioavailability of
VEGF in angiogenesis (Shchors et al.,
2006). IL1β mobilizes VEGF from the extracellular matrix
(ECM) to ECs during active angiogenesis, via its ability to promote expression
and proteolytic activation of stromal MMPs
(Mountain et al., 2007;
Shchors et al., 2006). MMPs
not only modulate the ECM but also cleave the ECM binding domain of VEGF and
release isoforms of VEGF to ECs (Bergers et
al., 2000). Depletion of these and other paracrine factors
originating from hematopoietic cells is likely to contribute to the impaired
angiogenesis in the mutant, despite the elevated VEGF mRNA levels.
We therefore suggest that a combination of defects including reduction in hemodynamic stress and hypoxia-induced apoptosis with a shortage in proangiogenic factors contributes to the vascular defects in the mid-gestation c-myc mutant embryos. If these vascular defects resulted exclusively from the loss of HCs, then preserving c-myc in HCs while deleting it in ECs should allow the mutant embryos to survive beyond midgestation and develop a normal vasculature. Indeed, when we deleted c-myc in the majority of ECs but only a subset of HCs using the Tie1-Cre line, all of these mutants survived to late gestation, well past the lethality of Vav-iCre;c-mycflox/- embryos. These results suggest that c-Myc regulates angiogenesis through its control over hematopoiesis and the production of paracrine factors.
The physiological function of c-myc may be restricted to hematopoietic lineages in the embryo and placenta
Retention of c-myc in the visceral endoderm and the
extra-embryonic ectoderm of Sox2-cre mutants prevented the gross
organ abnormalities seen in c-myc-/- embryos and extended
the embryo survival, demonstrating that c-myc plays an essential
function in these tissues. In the embryo proper, the severe Vav-iCre
CKs phenocopied the Tie2-Cre and Sox2-Cre CKs, suggesting
the possibility that c-myc in the HCs is most crucial for the
development and survival of the embryo at this stage.
Therefore, our genetic evidence suggests the possibility that
c-myc functions restrictively in placenta and HCs but less so in
other tissues. Supporting this notion, we found no significant cell autonomous
requirement for c-myc in ECs. Other studies also indicate that c-Myc
is dispensable for the homeostasis of the adult intestinal epithelium
(Benitah et al., 2005;
Oskarsson et al., 2006),
postnatal hepatocyte proliferation (Baena
et al., 2005) and liver regeneration
(Li et al., 2006). Although
further investigation is required to delineate the precise physiological
function of c-Myc, our data and the published findings raise the hypothesis
that c-myc may be uniquely required in the hematopoietic lineage and
placenta, playing a less crucial role in other cell lineages in vivo.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/14/2467/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: University of California, San Diego, Department of
Pathology, San Diego, CA 92103, USA
Present address: Institute for Diagnostic Imaging, Technical University
Munich, 81675 Munich, Germany
Present address: Estrada Control Nicolau de Mesquita, Hovione
PharmaScience, Taipa, Macau
¶ Present address: Abbott Vascular, Abbott Laboratories, Abbott Park, IL
60064, USA
|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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