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First published online May 9, 2008
doi: 10.1242/10.1242/dev.011296
1 Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN
38105, USA.
2 Department of Molecular and Cellular Pharmacology, University of Miami Miller
School of Medicine, Miami, FL, USA.
3 Department of Cell Biology, University of Miami Miller School of Medicine,
Miami, FL, USA.
4 Department of Cancer Biology, The Scripps Research Institute-Florida, Jupiter,
FL 33458, USA.
* Authors for correspondence (e-mails: jcleve{at}scripps.edu; mking{at}med.miami.edu)
Accepted 26 March 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: Myc, Slug/Snail2, Twist, Vasculogenesis, Lymphangiogenesis, Xenopus
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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;
Grandori et al., 2000). Under
normal conditions, the transcription of Myc genes is mitogen dependent and is
suppressed by growth inhibitory signaling pathways and by the tumor
suppressors p53 and pRb (Grandori et al.,
2000; Bernard and Eilers,
2006). The pervasive selection for Myc overexpression in cancer
reflects its pivotal roles in regulating progression through the cell cycle
(Roussel et al., 1991),
increases in cell mass (Iritani and
Eisenman, 1999) and the tumor angiogenic response
(Pelengaris et al., 1999;
Baudino et al., 2002;
Knies-Bamforth et al.,
2004).
Loss-of-function experiments have revealed a conserved role for Myc in
regulating cell growth and division in both vertebrates and invertebrates. For
example, in Drosophila mosaic dmyc (dm - FlyBase)
mutants, the wing discs grow very poorly, are smaller in size and are
out-competed by wild-type cells (Johnston
et al., 1999). Furthermore, the conditional knockout of
Myc in mice established its essential role in the G1- to S-phase
transition of the cell cycle (de Alboran
et al., 2001; Trumpp et al.,
2001). However, there are also obvious consequences of
Myc loss on developmental processes. For example, the conditional
deletion of Myc in the hematopoietic compartment leads to an
accumulation of hematopoietic stem cells that fail to migrate from the bone
marrow (Wilson et al., 2004)
and Myc loss in the mouse leads to stunted growth, neural tube
closure defects and pericardial swelling, and to profound defects in embryonic
vasculogenesis, angiogenesis and hematopoiesis
(Davis et al., 1993;
Trumpp et al., 2001;
Baudino et al., 2002).
The broad and devastating effects of Myc loss on mouse embryonic
development suggested that many might be attributed to hematopoietic failure,
and/or to defects of Myc-/- embryos in vasculogenesis
(Baudino et al., 2002). Indeed,
mouse embryos haploinsufficient for the angiogenic cytokine vascular
endothelial growth factor (Vegf) (Carmeliet
et al., 1996; Ferrara et al.,
1996) or its receptors Flt1 (VegfR1)
(Fong et al., 1995) or Flk1
(Kdr - Mouse Genome Informatics) (Shalaby
et al., 1995) display phenotypes that are similar to those
manifest by the Myc knockout. Furthermore, Myc is expressed in
endothelial-like progenitors of the blood islands that are known to give rise
to the primitive vascular and hematopoietic system, and Myc-deficient cells
have defects in the expression of angiogenic regulators
(Baudino et al., 2002).
Although the defects in vascular development in the mouse Myc
knockout suggested this was a cause of lethality, the failure of these embryos
could also be due to placental defects. Xenopus laevis is a tractable
model system that lacks this concern and allows the analysis of pathways that
control cell fate decisions during early embryogenesis. More importantly, the
vascular system of the Xenopus embryo is easily visualized and is
very similar to that of higher vertebrates
(Levine et al., 2003;
Ny et al., 2005). Furthermore,
these embryos can survive without a circulatory system for an extended time,
permitting mutants to be analyzed well into development
(Mohun et al., 2000). Indeed,
here we report that specific knockdown of Xc-Myc in post-gastrulation
stages does not lead to defects in embryonic hematopoiesis or vasculogenesis
per se, but rather leads to defects in the maturation and completion of vessel
development in both the vascular and lymphatic systems. Importantly, these
defects were rescued by the transcription factors Twist and/or Slug (also
known as Snail2), indicating that a Myc-Twist/Slug circuit is required to
direct and complete normal vessel development.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Knockdown experiments were performed by injecting embryos
at the one-cell stage with 40 ng of Xc-Myc morpholino (Mo). Control
morpholinos were injected at 40-80 ng per embryo. Following injections,
embryos were visually inspected at the indicated stages of development until
they reached stage 45, and were scored for the presence of edema. Embryos were
stained with benzidine (Hemmati-Brivanlou
and Thomsen, 1995) to visualize effects on Xc-Myc knockdown on
blood development and the vascular system. For rescue experiments, 500 pg of
Xc-Myc-Mut, XSlug or XTwist mRNA were mixed with 40 ng Mo
and injected at the one-cell stage. Injections of wild-type Xc-Myc or
β-galactosidase mRNA with Mo were used as controls. Embryos used
for in situ hybridization were injected at the two-cell stage, in one
blastomere, with half the dose of Mo (20 ng) or RNA (250 pg) mixed with mRNA
encoding β-galactosidase to mark the injected side. At least
three different batches of embryos obtained from different frogs were used for
each set of experiments.
Lineage analysis was initially carried out at the 32-cell stage, yet here
the morpholino dose proved toxic or failed to produce any phenotype at the
lower doses tested. Therefore, lineage analysis was done at the 16-cell stage
and provoked lineage-specific phenotypes. Following the fate maps of Moody
(Moody, 1987), blastomeres of
either the D2.1, V1.2 or V2.1 lineage were injected with a 1 nl mixture of
rhodamine-dextran and either Xc-Myc morpholino (800 nM), or a
combination of XSlug (266 nM) and XTwist (534 nM)
morpholinos. The embryos were cultured overnight and sorted at stage 13 based
on the presence of the rhodamine signal. Embryos which showed positive
labeling were followed until stage 45 and scored for developmental defects
including hemorrhage and edema. Lineage analyses were from different batches
of embryos obtained from four different frogs.
Western blot analyses, whole-mount in situ hybridization and histology
Whole embryo extracts were prepared by Freon extraction and western blots
were performed using a Myc antibody (Santa Cruz) that recognized Xc-Myc
protein. In situ hybridization and β-galactosidase staining were
performed according to standard methods
(Sive et al., 2000).
Digoxigenin-UTP-labeled antisense RNA probes were generated against
Xc-Myc, XTwist, XSlug and X-msr using MegaScript Kit
(Ambion). All results were collected from two to four different batches of
embryos obtained from different frogs.
For histology, staged embryos were fixed in MEMFA [0.1 M MOPS (pH 7.4), 2 mM EGTA, 1 mM MgSO4, 3.7% formaldehyde] overnight at 4°C, dehydrated in a graded ethanol series and embedded in paraffin. Sections (4 µm) were prepared and were stained with Hematoxylin and Eosin.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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),
Xenopus harbors two copies of Xc-Myc (Xc-Myc1 and
Xc-Myc2), both of which are maternally expressed. However, only
Xc-Myc1 is zygotically expressed following the mid-blastula
transition (Vriz et al., 1989)
(see Fig. S1 in the supplementary material). In the mouse embryo, Myc is
expressed in endothelial-like (PECAM-1+) cells that line the blood
islands of the yolk sac (Baudino et al.,
2002), which give rise to both the embryonic vasculature and
primitive hematopoietic cells. In Xenopus, the precursors of blood
and vasculature arise in an analogous region coined the ventral blood island
(Turpen, 1998). To define
precisely the spatial and temporal patterns of Xc-Myc expression in
Xenopus embryos, we performed whole-mount in situ hybridization
(Fig. 1A). The probe was
specific for Xc-Myc and does not recognize XN-Myc. During
gastrulation, Xc-Myc is expressed in the area flanking the yolk plug
corresponding to the involuting mesoderm
(Fig. 1A, part i). Furthermore,
during the neurula stages, Xc-Myc is expressed in the anterior,
posterior and lateral edges of the embryo, as previously described
(Bellmeyer et al., 2003). The
latter region harbors pre-migratory and migratory neural crest precursors
(Fig. 1A, parts ii,iii). At the
tailbud stage, Xc-Myc expression was evident in the developing brain,
eyes, somites, pharyngeal arches and, importantly, in the region corresponding
to the ventral blood islands (VBI, Fig.
1A, part iv). By stage 35/36, Xc-Myc expression expanded
into the region where the heart and outflow tract are formed and is strongly
expressed in the rostral lymph sac (RLS,
Fig. 1A, part v). Therefore, in
addition to its known expression and role in neural crest development,
Xc-Myc is also expressed in primitive mesoderm, in the VBI that gives
rise to the vascular system, and in the rostral lymph sac, a crucial component
of the lymphatic system.
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Knockdown of Xc-Myc did not lead to obvious external developmental
abnormalities through stage 28 (see Fig. S2 in the supplementary material),
although changes in gene expression were evident at this stage (see below,
Fig. 4B). As expected
(Bellmeyer et al., 2003),
Xc-Myc morpholino-injected embryos showed craniofacial defects by
stage 41 (Fig. 2A,
Fig. 3A). However, in addition,
we also observed obvious signs of ventral edema in Xc-Myc-knockdown embryos,
and by stage 45 this edema was profound
(Fig. 2A, white arrows).
Cross-sections of embryos at stage 37 revealed marked defects in the
development of the digestive tract that were associated with edema
(Fig. 2B), as well as defects
in the development of the somites and spinal cord, which were atrophied or
missing in Xc-Myc-knockdown embryos (Fig.
2B). Furthermore, at stage 37 the hearts of Xc-Myc knockdown
embryos lacked normal chambers (Fig.
2C) and large blood vessels such as the dorsal aorta and posterior
cardinal vein appeared extremely thin (Fig.
2D, Fig. 3C).
Therefore, knockdown of Xc-Myc in Xenopus leads to marked edema and
associated catastrophic effects on organogenesis.
Xc-Myc knockdown impairs endothelial cell assembly into patent tubules
We reasoned that many of the abnormalities observed during organogenesis
and the massive edema in Xc-Myc-knockdown embryos might reflect a defective
vasculature and/or lymphatic system. We ruled out one explanation for the
source of edema, incomplete closure of the neural tube, as we found this step
to be normal in Xc-Myc-knockdown embryos (data not shown). At the stages of
embryonic development affected, blood vessels are composed exclusively of
endothelial cells and pericytes, which are derived from mesoderm and neural
crest progenitors, respectively (Etchevers
et al., 2001; Cox et al.,
2006). To determine whether vascular endothelial cells were
correctly specified and/or assembled in Xc-Myc-depleted embryos, we assessed
the effects of Xc-Myc knockdown on the expression of the embryonic endothelial
lineage marker Xenopus mesenchyme-associated serpentine receptor
[X-msr (Devic et al.,
1996)].
Whole-mount in situ hybridization showed a reduced and more diffuse
staining pattern for X-msr in Xc-Myc knockdown stage 37 embryos
compared with uninjected embryos. This was especially evident in the blood
vessels of the head region, including the aortic arches and the anterior
cardinal vein, as well as in the vitelline vein network
(Fig. 3A,B). The vasculature
surrounding the lens (tunica vasculosa lentis) and the developing retina
(choroid) was also incomplete and/or collapsed
(Fig. 3A,B). One of the most
telling defects was in the rostral lymph sac (RLS), which appeared completely
missing. Normally this distinct structure, found just anterior to the heart
region, strongly expresses X-msr
(Fig. 3B)
(Ny et al., 2005). This sac is
composed of endothelial cells derived from a common endothelial precursor that
also gives rise to vascular progenitors originating in the lateral plate
mesoderm (Ny et al., 2005). In
Xc-Myc-depleted embryos X-msr staining is essentially absent in this
region (Fig. 3B, arrowhead).
Therefore, the Myc knockdown phenotype also includes defects in lymphatic
vessel development.
In most cases, the endothelial cells detected by X-msr expression in Xc-Myc knockdown embryos appeared to align in a roughly normal vascular pattern, yet their staining was much weaker and was often diffuse (Fig. 3A,B). Indeed, this was confirmed by detailed histological analyses of the posterior cardinal vein and the dorsal aorta, which revealed that endothelial linings of these major blood vessels were very thin and appeared to be composed of fewer endothelial cells (Fig. 3C).
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). Benzidine staining revealed that tadpoles injected with
control morpholinos had a well-developed vasculature, including the aortic
arches emanating from the heart (Fig.
4A). In Xc-Myc-knockdown tadpoles, blood was also evident;
therefore, there are no obvious effects of Xc-Myc loss on erythrocyte
development. However, the blood was pooled and localized in hemorrhagic areas
in different regions of the body proper in Xc-Myc-depleted embryos, most often
in the abdominal cavity (Fig.
4A). Moreover, in most of the Xc-Myc-knockdown embryos, the aortic
arches and peripheral vessels in the tail were not stained by benzidine,
indicating that blood had leaked from the vasculature. Indeed, quantification
of these phenotypes demonstrated that 
85% of Xc-Myc
morpholino-injected stage 45 embryos had profound edema
(Fig. 5A, n=339) and
that nearly all of these (n=316) had associated defects in vessel
development (Fig. 5B, part ii;
Fig. 5C), clearly linking these
two events. By contrast, fewer than 5% of uninjected embryos (n=617)
or embryos injected with a scrambled control Xc-Myc morpholino
(n=190) showed edema and vascular defects
(Fig. 5A,B, part i;
Fig. 5C). Defects in vascular development in Xc-Myc-knockdown embryos were observed well before edema became obvious at stages 40/41. For example, athough blood could be observed throughout the circulatory system of control embryos, it was largely absent in the peripheral circulatory system of Xc-Myc morpholino-injected stage 37 embryos (Fig. 3B).
To confirm that the developmental defects provoked by the Xc-Myc morpholino were not due to off-target effects, embryos were co-injected with the Xc-Myc morpholino and mRNA encoding a wobble mutant of Xc-Myc (Xc-Myc-Mut) designed to block its recognition by the Xc-Myc morpholino. Indeed, in vitro translation of Xc-Myc-Mut protein was unaffected by the Xc-Myc morpholino (Fig. 1B). Importantly, all of the defects that were manifest in Xc-Myc knockdown embryos, including edema (n=327) and vascular defects (n=240), were rescued by co-injection of mRNA encoding the mutant of Xc-Myc (Fig. 5A,B, part iii; Fig. 5C).
In addition to ameliorating the edema and vascular phenotypes of Xc-Myc
knockdown embryos, overexpression of Xc-Myc-Mut often led (in 55% of injected
embryos) to abnormal patterns of blood distribution, which were characterized
by ectopic vascular beds and hypervascularization
(Fig. 5B, part iii, black
arrows; Fig. 5C, red bars,
n=240). Furthermore, 70% of embryos injected with wild-type
Xc-Myc mRNA alone displayed an ectopic formation of blood vessels
(Fig. 5B, part iv, black
arrows; Fig. 5C, red bars,
n=146), consistent with findings in mice that have demonstrated Myc
overexpression can provoke angiogenesis
(Pelengaris et al., 1999;
Knies-Bamforth et al., 2004).
Therefore, physiological thresholds of Myc are essential for normal formation
of patent vessels during vasculogenesis.
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; Carmeliet,
2003; Cleaver and Melton,
2003; Cleaver,
2004). In addition, we assessed the expression of X-erg,
an Ets family transcription factor that is expressed in vascular endothelial
cells, in the neural crest-derived mesenchymal cells of pharyngeal arches, and
in the endocardium. X-erg is thought to direct cell migration during
vascular development (Remy and Baltzinger,
2000; Tahtakran and Selleck,
2003). Although Vegf, Flk1 and Tie2 expression
changed little in Xc-Myc knockdown embryos, X-erg expression was
reduced by 50% (Fig.
4B).
Additional transcriptional regulators of vasculogenesis and
lymphangiogenesis include the transcription factors Scl, which plays critical
roles in yolk sac erythropoiesis and in angiogenic remodeling of the yolk sac
capillaries into complex vitelline vessels
(Mead et al., 1998), and
Prox1, a master regulator of lymphangiogenesis
(Wigle and Oliver, 1999;
Wigle et al., 2002). Notably,
knockdown of Xc-Myc led to marked reductions in the expression of both
Scl and Prox1 (Fig.
4B). Like X-erg, reduced levels of Scl and Prox1 would be
predicted to contribute to the edematous phenotype of Xc-Myc knockdown
embryos, which displayed marked defects in the ventral vascular plexus and
rostral lymph sac.
A Myc-to-Slug/Twist pathway directs vasculogenesis and lymphangiogenesis
Knockdown of Myc in Xenopus results in the loss of neural crest
derivatives, such as craniofacial structures, cartilage and fins, and
compromises the expression of the Slug and Twist
transcription factors in the neural crest. Indeed, Slug and
Twist are also important for cell fate determination and migration of
the neural crest and function downstream of Xc-Myc
(LaBonne and Bronner-Fraser,
2000; Bellmeyer et al.,
2003). However, Slug and Twist are also
expressed in the lateral plate mesoderm, which generates endothelial precursor
cells (Hopwood et al., 1989;
Mayor et al., 1995). This
suggested that Slug and/or Twist might also function downstream of Xc-Myc to
coordinate normal blood and lymph vessel development. As expected, injection
of Xc-Myc morpholino into one cell of a two-cell embryo disrupted the
expression of Twist and Slug on the injected side, and these
defects were rescued by the co-injection with the mRNA encoding the wobble
mutant of Xc-Myc (see Fig. S3 in the supplementary material). Furthermore,
co-injection of Xc-Myc morpholino and Slug or Twist
mRNA into one cell of two-cell embryos rescued defects in endogenous
XSlug or XTwist expression (see Fig. S3 in the supplementary
material). Therefore, either XSlug or XTwist can function
downstream of Myc.
To test the potential functions of XSlug or XTwist downstream of Myc in vessel development, one-cell embryos were co-injected with Xc-Myc morpholino and Slug or Twist mRNAs, and assessed at stage 45 by staining with benzidine. Notably, many embryos injected with Xc-Myc morpholino together with Slug (n=181) or Twist (n=242) mRNA did not develop edema or hemorrhage, a hallmark of those injected with the Xc-Myc morpholino alone (Fig. 6A,B, n=316). Rescue of this phenotype was specific for Slug or Twist mRNA, as embryos co-injected with the Xc-Myc morpholino and β-galactosidase mRNA (n=169) displayed profound defects in vascular development (Fig. 6A). Indeed, Slug or Twist mRNA/Xc-Myc morpholino co-injected embryos (n=327) were phenotypically similar to normal embryos and to those rescued by co-injection of the wobble Xc-Myc-Mut mRNA (n=108, Fig. 6A,B). However, Slug- or Twist-injected embryos lacked the hyper-vascularization phenotype that characterized Xc-Myc overexpression (Fig. 5B,C). Therefore, enforced expression of either Slug or Twist can specifically rescue the defects in vessel development provoked by Xc-Myc knockdown.
To investigate more directly what cell lineage Xc-Myc, XTwist and XSlug
were acting through to affect vessel development, we injected their
corresponding morpholinos together with a lineage tracer into blastomeres of
the 16-cell stage embryo whose descendants mostly contribute to either neural
crest or lateral plate mesoderm. Both XTwist
(Hopwood et al., 1989) and
XSlug (Mayor et al., 1995) are
expressed in lateral plate mesoderm. The V1.2 blastomeres are a major
contributor to both trunk and head neural crest, whereas D2.1 mostly to head
neural crest and lateral plate mesoderm. V2.1 contributes mostly to lateral
plate mesoderm and nothing to head neural crest
(Fig. 7A,B)
(Moody, 1987). Specifically,
descendents of the D2.1 lineage give rise to embryonic blood, endocardium and
endothelial cells that comprise the ventral aorta and vitelline veins, whereas
V2.1 descendents give rise to blood precursor cells and endothelial cells of
major blood vessels (Walmsley et al.,
2002). Notably, Xc-Myc or XSlug/XTwist knockdown within the neural
crest lineage (V1.2 or D1.2) did not result in edema (0%, n=41; 0%,
n=29) and very few (3%, n=41; 7%, n=29) developed
hemorrhagic spots (compare Fig.
7B,C, V1.2, controls with Fig.
4A). In sharp contrast, Xc-Myc morpholino injections into D2.1
(lateral plate mesoderm) resulted in 44% edematous and 54% hemorrhagic embryos
(n=57, Fig. 7B).
Similar results were obtained for the XSlug/XTwist MO D2.1 injections (33%,
33%, n=18) with most embryos displaying both phenotypes
(Fig. 7B,C). Interestingly,
knockdown of Xc-Myc or XSlug/XTwist in the posterior lateral plate mesoderm
(V2.1) was sufficient to cause edema in the head (33%, n=15) and
edematous or hemorrhagic areas throughout the embryo (66%, n=15; and
75%, n=12). These findings are inconsistent with a cell-autonomous
effect of Xc-myc, XSlug or XTwist in the neural crest lineage for vascular
development. Furthermore, a lack of benzidine staining of the brachial arches,
heart and outflow tract was also observed in the posterior lateral plate
mesoderm targeted knockdowns (Fig.
7B,C). Collectively, these findings show that Xc-Myc and
XSlug/XTwist operate in a regulatory pathway outside of the neural crest
lineage, but within the lateral plate mesoderm, to affect both the endothelial
and blood lineages.
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Wigle and Oliver, 1999;
Wigle et al., 2002;
Tahtakran and Selleck, 2003).
Furthermore, Slug and Twist, transcription factors that function downstream of
Myc in neural crest development (Bellmeyer
et al., 2003), are shown here to also function downstream of Myc
in directing normal vessel development within the lateral plate lineage, a
site where both genes are also expressed
(Mayor et al., 1995;
Hopwood et al., 1989). Given
the observed effects of knockdown of Slug on the expression of
Xc-Myc, the ability of Slug or Twist to rescue the phenotypes
manifest following Xc-Myc knockdown, and the ability of XSlug/XTwist
morpholinos to cause vascular defects similar to Xc-Myc knockdowns, these
findings support a model whereby Myc, Twist, and Slug function in a regulatory
circuit that also directs the maturation of vessels of the vascular and
lymphatic systems (Fig. 8).
Myc is required for normal vessel development in Xenopus
In the mouse, Myc appears required for primitive erythropoiesis and
vasculogenesis (Baudino et al.,
2002). In our studies, Xc-Myc was not fully depleted until the
neural tube stage (stage 21), leaving open the possibility for an earlier role
for Xc-Myc in the specification of endothelial progenitors. Maternal Xc-Myc is
present at high levels in the unfertilized egg and could provide a source of
this protein to the early embryo (Vriz et
al., 1989). However, loss of Xc-Myc by stage 21 provokes massive
hemorrhage and edema, which are indicative of vascular defects.
Xc-Myc-depleted embryos displayed very thin vessels that were generally devoid
of blood cells, which apparently had leaked out and pooled in the embryo body
proper. These phenotypes were intrinsic to Myc. As progenitors for blood and
endothelial cells first appear just posterior to the cement gland and in the
lateral plate mesoderm at the neurula stages
(Walmsley et al., 2002;
Cleaver, 2004), we infer that
these progenitors, and the lymphangioblasts and endothelial cells that arise
from them (Ny et al., 2005),
require Xc-Myc to mature and form patent fully functional vessels. Conversely,
we also infer that the hypervascularization phenotype observed in embryos
injected with Xc-Myc mRNA may be due to increases in numbers of
angioblasts and/or their progeny, which results in the production of excess
vessels, a phenotype that is akin to those observed following ectopic
expression of Vegf in Xenopus
(Cleaver, 2004).
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). By contrast, in Xenopus, in situ
hybridization and histological analyses revealed that many of the early steps
in vasculogenesis occurred normally in Xc-Myc knockdown embryos, including the
specification of endothelial cell precursors (angioblasts), their migration
and coalescence into continuous strands of endothelial cells at correct
locations in the embryo, and proper expression of Vegf and
Flk1. Thus, given that Xc-Myc is expressed in the lateral plate
mesoderm and VBI where angioblasts form, these developmental steps are either
independent of Xc-Myc, or occur before effective MO Xc-Myc knockdown at stage
21.
Xc-Myc is required in endothelial cells for vessel maturation
Xc-Myc may regulate Xenopus vascular development at different
levels. First, the numbers of endothelial cells may be reduced in Xc-Myc
knockdown embryos, suggesting at least a partial role for Xc-Myc in the early
expansion of this cell lineage. Interestingly, Bellmeyer et al.
(Bellmeyer et al., 2003) ruled
out a role for Xc-Myc in proliferation within the neural crest lineage.
Although we were unable to detect obvious differences in cell proliferation
after knockdown of Xc-Myc using histone markers for proliferation (data not
shown), Xc-Myc has been reported to regulate cell proliferation in
Xenopus (Etard et al.,
2005). Second, as endothelial cells assemble into tubes later in
development, they may fail to form normal intercellular junctions following
Xc-Myc knockdown, resulting in inadequate sealing. Third, smooth muscle cells
or pericytes may fail to recruit to endothelial tubes, which would also result
in leaky vessels. The latter hypothesis was appealing given the involvement of
Myc in neural crest specification in Xenopus
(Bellmeyer et al., 2003) and
the contribution of neural crest cells to smooth muscle and pericytes in the
anterior regions of the cardiovascular system
(La Bonne and Bronner-Fraser,
1999; Huang and Saint-Jeannet,
2004). However, our data support endothelial cells as the crucial
target as we observed leaky vessels devoid of red blood cells by stage 37, a
time prior to the recruitment of smooth muscle cells and pericytes to vessels
(Warkman et al., 2005;
Cox et al., 2006). In
addition, Xc-Myc-depleted embryos lacked a distinct rostral lymph sac
(Fig. 3B), a structure
comprised only of endothelial cells that are held together by desmosomal-like
structures (Ny et al., 2005).
Collectively, these observations are therefore most consistent with
Xc-Myc-depleted embryos being defective in endothelial numbers and cell-cell
adhesion, rather than in mural cell recruitment. Together with our lineage
studies, these results further support a role for Xc-Myc in vascular
development independent of the neural crest. However, as neural crest cells
migrate over the mesoderm, we cannot rule out that perturbing mesodermal
lineages does not also affect neural crest migration, although we can rule out
a cell-autonomous effect of c-Myc on the neural crest lineage in vascular
development.
The Myc-Slug/Twist regulatory circuit
Slug and Twist perform important developmental roles as mediators of the
epithelial-to-mesenchymal transition by regulating the expression of
cell-adhesion molecules such as V-cadherin
(Bolos et al., 2003;
Marin and Nieto, 2004), which
are also regulated indirectly by Myc through Slug
(Wilson et al., 2004). More
recently, Twist and Slug have been suggested to play crucial roles in cancer
and tumor angiogenesis, processes that are also regulated by Myc
(Pelengaris et al., 1999;
Baudino et al., 2002;
Valsesia-Wittmann et al.,
2004; Elloul et al.,
2005; Yang et al.,
2006). Slug and Twist have well described roles in neural crest
development in Xenopus but have never been associated with vascular
development in this organism (LaBonne and
Bronner-Fraser, 1999; LaBonne
and Bronner-Fraser, 2000;
Huang and Saint-Jeannet,
2004). Notably, Xc-Myc knockdown disrupted the normal expression
of Slug and Twist (data not shown), and ectopic Twist or
Slug were able to rescue the vascular defects of Xc-Myc knockdown embryos.
Moreover, Slug knockdown affected the expression and/or migration of
Xc-Myc-expressing cells, indicating a feedback loop in this
regulatory circuit (Fig. 8).
However, knockdown of XSlug alone did not elicit vascular defects, but an
XSlug/XTwist dual knockdown did reveal redundancy in this pathway
(Fig. 7B,C). Collectively,
these findings support a model whereby Myc, Twist and Slug also function in a
regulatory circuit that directs vasculogenesis and lymphangiogenesis outside
the neural crest lineage (Fig.
8).
We propose that endothelial cell-cell interactions are directed by Xc-Myc.
Also consistent with this model, we found that Xc-Myc depletion compromised
the expression of Scl, which directly induces the expression of
vascular endothelial (VE)-cadherin
(Deleuze et al., 2007).
Notably, VE-cadherin is an essential adhesion factor at intercellular
junctions important in the patency of blood and lymph vessels
(Ny et al., 2005). Therefore,
Myc, together with Slug and/or Twist, may regulate the assembly of blood
vessels by directing the expression of these crucial adhesion receptors.
Future studies will explore these models of Myc function.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/11/1903/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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