1 Department of Physiology, Cardiovascular Research Institute Maastricht
(CARIM), Maastricht University, The Netherlands
2 Inserm U36, Collège de France, 11, Place Marcelin Berthelot, 75005
Paris, France
3 Department of Anatomy, University of Bern, Switzerland
4 Department of Physics, Ecole Polytechnique, Palaiseau, France
* Author for correspondence (e-mail: anne.eichmann{at}college-de-france.fr)
Accepted 17 October 2003
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SUMMARY |
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Key words: Arterial-venous differentiation, Hemodynamic forces, Growth factors, Receptors, Ephrin, Neuropilin
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Introduction |
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During development, blood vessels in the embryo and in the yolk sac are
initially formed by a process termed vasculogenesis, which denotes the in situ
differentiation of EC from the mesoderm and their coalescence into primary
vessels (Risau, 1997).
Vasculogenesis leads to the formation of the first major intra-embryonic blood
vessel, the aorta, and to the formation of the primary vascular plexus in the
yolk sac. With the onset of embryonic circulation, these primary vessels have
to be remodeled into arteries and veins, in order to develop a functional
vascular loop and to accommodate cardiac output. Remodeling of the primary
vascular plexus into a more mature vascular system is thought to occur by a
process termed angiogenesis. Angiogenesis in the yolk sac involves capillary
sprouting, splitting and remodeling, which leads to the reorganization of the
primary vessels into large and small vessels
(Risau, 1997
). Surprisingly,
the formation of yolk sac arteries and veins has not been specifically
addressed in the recent literature.
EC were long considered as a homogenous population of cells
(Risau, 1997), and
differentiation of arteries and veins was thought to be governed by
hemodynamic forces, molding out these vessels from the primary vascular
plexus. This idea was based on classic studies carried out by Thoma in the
chick embryo yolk sac (Thoma,
1893
). Thoma observed that vessels that carry a lot of blood flow
widen, while those that carry little flow regress. Murray subsequently
postulated that vessels adapt to flow in order to optimize the shear stress to
which they are subjected (Murray,
1926
). These studies have shown that flow can alter lumen
dimensions of arterial segments. However, they did not address if or how flow
contributes to arterial-venous differentiation and patterning. Studies
subsequently carried out in adult vessels showed that vessel segments can
adapt to the amount of flow carried
(Peirce and Skalak, 2003
;
Skalak and Price, 1996
). Flow
can alter the expression and release of growth factors implicated in vascular
remodeling including nitric oxide, endothelin 1, FGF and PDGF.
Recently, specific markers for arteries and veins were discovered, which
labeled EC from early developmental stages onwards, before the assembly of a
vascular wall. For the arterial system, ephrinB2, neuropilin 1 (NRP1), and
members of the Notch pathway, including notch 3, Dll4 and gridlock have been
described in zebrafish, chick and mouse
(Herzog et al., 2001;
Lawson et al., 2001
;
Moyon et al., 2001a
;
Moyon et al., 2001b
;
Shutter et al., 2000
;
Villa et al., 2001
;
Wang et al., 1998
;
Zhong et al., 2001
). Other
molecules are specifically expressed in the venous system, most notably EphB4,
the receptor for arterial ephrinB2 (Gerety
et al., 1999
). The neuropilin 2 (NRP2) receptor is expressed by
veins and, at later developmental stages, becomes restricted to lymphatic
vessels in chick and mice (Herzog et al.,
2001
; Yuan et al.,
2002
). In chick, the Tie2 receptor is expressed at higher levels
in veins compared with arteries (Moyon et
al., 2001b
). Based on these specific expression patterns and on
mutant studies in zebrafish and mouse, it has been suggested that
arterial-venous differentiation during embryogenesis is actually genetically
predetermined (Wang et al.,
1998
). Interestingly, ephrins and neuropilins are also expressed
in the developing nervous system where they are implicated in the
establishment of cell boundaries and axon guidance
(Flanagan and Vanderhaeghen,
1998
). It is tempting to speculate that the growth control
processes in the nervous system, exerted through repulsive and attractant
signaling, may also apply to the vasculature
(Kullander and Klein,
2002
).
Mouse mutants for ephrinB2 and EphB4 die at embryonic day (E) 9.5 because
of defective remodeling of the primary vascular plexus into branched arteries
and veins. The specific effects on arteries and veins were recognized because
of the restricted expression of these molecules
(Adams et al., 2001;
Gerety et al., 1999
;
Wang et al., 1998
).
Interestingly, phenotypes of mouse mutants defective for the ligand (ephrinB2)
and the receptor (EphB4) are symmetrical. Both molecules are
transmembrane-spanning, raising the question of how and where they interact
during the formation of arteries and veins. Moreover, both ligand and receptor
are able to elicit a signaling cascade. In the nervous system, this
bi-directional signaling is involved in the formation of cell boundaries in
the rhombomeres (Klein, 1999
).
In the vascular system, it has been shown that reverse signaling initiated by
EphB4 binding to its ligand ephrinB2 is responsible for the observed
remodeling failure (Adams et al.,
2001
). Many other mouse mutants for genes involved in vascular
development die because of failure in vessel remodeling, including
angiopoietin 1 (Suri et al.,
1996
), HIF1
(Ryan et
al., 1998
; Iyer et al.,
1998
), VE-cadherin (Carmeliet
et al., 1999
), activin receptor-like kinase 1
(Urness et al., 2000
) and
connexin 45 (Krüger et al.,
2000
), as well as signaling molecules specifically expressed by
arteries, such as NRP1 and members of the Notch family
(Kawasaki et al., 1999
;
Swiatek et al., 1994
;
Xue et al., 1999
). In these
latter mutants, a specific effect on arteries has not been described, rather a
general failure to form large and small vessels in the yolk sac has been
noted.
It remains currently unknown if the remodeling of the primary vascular
plexus into arteries and veins is governed by flow, as suggested by the
classic studies (Murray, 1932; Thoma,
1893), or genetically predetermined, as suggested by the early
expression of arterial and venous markers and zebrafish studies. Zhong et al.
(Zhong et al., 2001
) have
shown that individual fluorescent angioblasts give rise to arterial or venous,
but not mixed clones, which is suggestive of a genetic predetermination.
Inactivation of the Notch pathway by dominant-negative expression of
suppressor of Hairless led to ectopic expression of venous markers in arteries
(Lawson et al., 2001
),
suggesting that the Notch pathway is required to suppress venous fate in
arterial endothelium. In spite of a possible genetic predetermination, two
recent studies reported that EC can still become incorporated into the venous
system after acquiring the expression of the arterial-specific genes ephrinB2
or NRP1 (Moyon et al., 2001a
;
Othman-Hassan et al., 2001
).
These studies suggest that EC may be genetically programmed for a certain
phenotype but can display plasticity with respect to local cues, probably
hemodynamics, the vascular wall or oxygen.
The aim of this study was to explain the regulation of the morphogenesis of the arterial and venous system with respect to the activation of genes specifically implicated in arterial-venous differentiation. We show that arterial-venous differentiation in the chick yolk sac is achieved after the onset of blood flow, and requires endothelial plasticity. We describe four crucial steps for the formation and patterning of arteries and veins resulting in paired and interlaced arteries and veins.
To achieve this, the disconnected segments make sprouts growing dorsal to arteries, projecting to the primary veins. Thus, vessels originally part of the arterial tree contribute to the formation of the venous tree. This secondary venous plexus now lies dorsally to the arteries and EC of both vessel types are now in a cis-trans configuration. The process of disconnection and reconnection allows the formation of paired and interlaced arteries and veins. Expression of the arterial markers ephrinB2 and NRP1 is regulated by blood flow and becomes rapidly downregulated in the disconnected segments. Instead, the plexus located dorsal to the arteries starts to express the venous markers NRP2 and Tie2, genetically confirming their venous identity. Similarly, experimental manipulation of the flow pattern changed global patterning of the yolk sac arteries and veins and regulated expression of arterial and venous markers accordingly.
The observation that ephrinB2 and NRP1 were regulated by flow prior to the subsequent changes in the vascular system suggested that they might be `markers' instead of `makers'. To test their function, we applied recombinant proteins in the yolk sac and allantois of chick embryos. Ephrin B2 and EphB4 application on yolk sac vessels failed to induce any obvious morphological changes of arteries and veins. By contrast, application on more mature arteries and veins in the allantois induced the formation of arterial-venous shunts. These results suggest that ephrinB2/EphB4 interaction may play a role in the maintenance of the mature vessel configuration rather than in its establishment.
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Materials and methods |
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Flow manipulation: no-flow and ligation model
To generate no-flow embryos, three different approaches were used to
obliterate the formation of a functional loop between heart and yolk sac.
Similar results were obtained with all three methods. In the first method, we
excised the head at the 10 ss, which effectively impeded formation of inflow
to the heart. In the second method we excised the heart at the 13-14 ss. The
third method mainly used in this work consists of slight dehydration of the
embryo, generated by opening a window in the egg-shell prior to incubation.
The egg is then sealed with Scotch tape into which holes are made and
incubated at 38°C until the desired stage.
Ligation of the right vitelline artery was performed using a tungsten wire
tool (0.5 mm diameter) as previously described
(Stephan, 1952). Briefly, the
tool (see Fig. 8) contains one
extending part, which is introduced underneath the artery and subsequently
lifts it. This lifting results in a mechanical obstruction of the artery
lumen, which blocks flow through the artery. Unmanipulated, age-matched
embryos were used as controls.
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In vivo application of ephrinB2-Fc and EphB4-Fc protein
Recombinant ephrinB2-Fc and EphB4-Fc protein were dissolved in sterile PBS,
and applied at a final concentration of 2 µg or 4 µg of protein per 5
µl of PBS. For treatment of the yolk sac in ovo, the vitelline membrane was
removed and protein was applied into a silicon ring placed on the yolk sac.
For treatment of New cultures, proteins were applied without rings. For
treatment of the allantois membrane, protein was applied within a silicon ring
at E4 (90 hours incubation). Application on older embryos had no effect.
Pre-clustering of ephrinB2-Fc or EphB4-Fc was achieved by pre-incubation with
anti-Fc antibody (rabbit anti-human IgG, Fc fragment specific, Jackson
ImmunoResearch, USA) at a concentration ratio of 1:8, for 1 hour at
4°C.
Time-lapse video-microscopy
New cultures were prepared as previously described
(Chapman et al., 2001). For
time-lapse video-microscopy, embryo New cultures or eggs containing a window
(LeNoble et al., 1993
), were
placed between two glass heating plates (Minitüb HT300, Germany), the
lower one at 37°C, the upper one at 39°C to avoid condensation.
Embryos were observed using a Leica MZFLIII stereo-microscope equipped with a
digital camera (Princeton Coolsnap cf). Images were acquired using Metaview
software (Princeton, version 5.0r6, 2002, US).
An intravital microscope setup (Leica) was used to obtain vessel images at
a high magnification and high resolving power. The vasculature was visualized
with a 4x (N.A.=0.12) or 11x objective (N.A.=0.25) using polarized
epi-illumination from a 100W Hg lamp. Images were projected onto a Hamamatsu
camera attached to an intensifying unit. The camera was connected to a video
recorder (Panasonic) and images were stored on S-VHS tape for off-line
analysis of vessel dimensions, and blood flow characteristics
(Rouwet et al., 2000).
In situ hybridization
In toto in situ hybridization using antisense mRNA probes for ephrinB2 and
NRP1 were performed as described previously
(Moyon et al., 2001a). 2114 bp
of coding sequence of chick NRP2 (Accession Numbers, AF417235.1 and AF417236)
were amplified by RT-PCR using two primers (forward, CTC AAC TTC AAC CCT CAC
TTC; reverse, ATC CGG TAC TCC ATG TCG TAG) from E8 chick embryo RNA. The
fragment was subcloned into T/A TOPOII vector (Invitrogen).
DiI-ac-LDL, FITC-dextran, QH1 and TUNEL staining
DiI-ac-LDL (Biomedical Technologies 200 µg/ml in PBS) and FITCDextran
(Sigma, Mr 2000 kDa, 8 mg/ml in PBS) were injected
intracardially using a micropipette. Embryos were observed immediately (Fitc
dextran) or after a 2 hours re-incubation period (DiI-Ac-LDL). TUNEL stainings
were done on paraffin sections (7.5 µm) prepared from chick embryos at the
25 and 35 ss, using an in situ cell death detection kit with peroxydase (Roche
Diagnostics) according to the manufacturer's instructions. Whole-mount QH1
stainings were done as previously described
(Pardanaud et al., 1987),
using undiluted hybridoma supernatant and GAM IgM coupled to Texas Red
(Southern Biotechnology Associates).
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Results |
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Theoretically, another possibility for the existence of the blood-filled
`spots' is that there may be degenerating vascular segments through apoptosis,
as previously suggested (Risau,
1997). To test this possibility, we sectioned yolk sacs at these
stages and examined apoptosis of EC by TUNEL staining. We could not detect any
significant number of apoptotic EC in yolk sac blood vessels, while some
endodermal cells showed TUNEL staining (not shown). Thus, pruning of the
vitelline arterial tree and remodeling of the veins may not involve
apoptosis.
In summary, we have observed that during vitelline artery development, small arteriolar side-branches are selectively disconnected and broken off the arterial tree. These disconnected vessels may not carry flow for a period of 5-6 hours, after which, through a process of directed sprouting, they reconnect to the primary venous system, and are again perfused. Thus, the separation of the arterial and venous system is achieved by breakage whereas the venous system grows by incorporating previously existing small arterioles. During normal development, EC plasticity thus seems to be an essential requirement for the growth of the veins.
During this process the system evolves from a two-dimensional architecture allowing cis-cis interactions between EC, the primary circulation, to a three-dimensional architecture, the secondary circulation, which allows cis-trans interaction between EC of the arterial and venous system, respectively. Mercox casts clearly showed that the vitelline artery comes to lie ventrally to the capillary plexus. This transition from a two-dimensional system to a three dimensional system with a dorsoventral polarity, is not restricted to chick embryos, it can also be observed in mouse embryo yolk sac during the period E10-E13 (data not shown). In fact, the patterning of the mouse and chick embryo yolk sac vessels show marked similarities.
Manipulations of blood flow and the effects on arterial-venous differentiation and patterning
No flow model
In the absence of perfusion in no-flow embryos (see Materials and methods),
the yolk sac develops a distinct vascular plexus, which contains blood islands
filled with erythrocytes in all embryos
(Fig. 7). This plexus can
continue to grow until at least E7 in the absence of perfusion. In spite of
this growth, its morphology continues to resemble the yolk sac of a 13-15 ss
embryo and no signs of arterial-venous differentiation are detected
(Fig. 7A). In situ
hybridization with ephrinB2 shows that part of the no-flow yolk sac vascular
plexus expresses this gene, whereas other regions are clearly negative
(Fig. 7B,C). The initiation of
arterial marker expression does thus not depend on perfusion of the yolk
sac.
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To further test this idea, we performed ligation experiments of the already formed vitelline artery. Ligation of the right vitelline artery was achieved by introducing a metal clip in ovo underneath the artery, the clip was devised such that it gently lifts the vitelline artery and arrests blood flow distal to the clip without perturbing general embryonic development. Embryos treated in this way can survive at least until E8, they do not exhibit overt morphological abnormalities and can develop a chorioallantoic membrane. We routinely incubated ligated embryos for 3 days and found morphology to proceed normally, except for the changes in the vascular patterning of the yolk sac described below.
Immediately after the ligation of the right vitelline artery, flow was attracted from the marginal vein and, hence, part of the arterial system was perfused in a retrograde manner. In 100% of the embryos (n=70), ligation resulted within 24 hours in complete venularization of the right side of the yolk sac (Fig. 8, Movie 4 at http://dev.biologists.org/supplemental). We observed that many previously existing arterial segments were incorporated into the new venular tree (compare Fig. 8B with 8I-L). The chance of a previously existing arterial segment to be incorporated into the venous tree appeared dependent on its orientation relative to the new preferential flow direction. Vessel segments oriented in the preferential flow direction (Fig. 8B,I-L) became incorporated into the venular tree. Other segments located immediately downstream of the ligation, more perpendicular to the new preferential flow direction, were not incorporated into the new venous tree but remained visible as unperfused tubes.
The ligation also affected growth of the left vitelline artery, and the anterior and posterior vitelline veins (Fig. 8A). About 24 hours after the ligation, large arteries branching off the left vitelline artery were observed to project to the right side of the yolk sac, and crossed the midline of the embryo/yolk sac axis (Fig. 8A, Fig. 9I,J), thus crossing the areas normally occupied by the anterior and posterior vitelline veins. Detailed examination of the fate of the anterior vitelline vein after ligation showed that it was remodeled and became partly integrated into the arteries that expand from the left side (Fig. 8A, Movie 5 at http://dev.biologists.org/supplemental), indicating that originally venous EC can be transformed into arteries. Thus, manipulation of the flow pattern can transform arteries into veins and veins into arteries.
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The downregulation of arterial marker expression could also be due simply to degeneration of the vessels. To test for this possibility, we first injected Fitc-dextran intravenously into embryos at different time points after ligation. Four hours after ligation, fluorescent signals could be readily detected in all vessels both on the unligated and on the ligated side (Fig. 10A). Time-lapse video-microscopy showed that perfusion of vessels on the ligated side originated from the peripheral sinus vein and the flow direction in the proximal part of the ligated vitelline artery was now reversed (see Movie 6 at http://dev.biologists.org/supplemental). No bleedings or plasma extravasations were detected, indicating that the experimental manipulation had not disturbed vessel integrity. We next performed ligations for different periods of time (30 minutes-8 hours), after which the ligation tools were removed and arterial flow was allowed to re-perfuse the right vitelline artery. The embryos were fixed 6 hours after removal of the ligation tools and hybridized with ephrinB2 (not shown) or NRP1 (Fig. 10B,C,F). Removal of the ligation tools after 30 minutes or 4 hours resulted in the re-expression of NRP1 mRNA in arterial vessels on the ligated side (Fig. 10B,C). Expression levels of NRP1 on the ligated side after 4 hours of ligation appeared low compared with the unligated side (Fig. 10C). However, in vivo examination of the right vitelline arterial tree after this 4 hour period of ligation followed by 12 hours of reperfusion revealed that the arterial tree on the ligated side was much smaller compared with the control side (Fig. 10D,E). Indeed, during the period of ligation, all the arterial blood flow passed through the left vitelline artery, which enlarged, allowing faster growth in comparison with the right side. After reperfusion, the growth of the right vitelline artery is therefore delayed (Fig. 10D,E). Reperfusion thus completely restores arterial marker expression, albeit in a reduced territory. After 6 hours of ligation, reperfusion was no longer obtained because of definitive obstruction of the vitelline artery and re-expression of arterial markers was not observed (Fig. 10F).
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We next examined the in vivo effects of ephrinB2 and EphB4 in the allantois membrane vessels at E4. Proteins were applied into silicon rings placed onto the allantois. Control application of the vehicle PBS showed no effects on vessel patterning (not shown). Strikingly, ephrinB2 overexpression in the allantois induced major changes in the vascular patterning (Fig. 11). In 5/5 embryos, the first changes in vessel patterning were observed several minutes after application and concerned veins, which appeared to increase the number of their branches. This was rapidly followed by the enlargement of both veins and arteries (compare Fig. 11A with 11I). Some arteries displayed a huge enlargement and increased their size more than 10-fold when compared with their initial diameter. Concomitant to the diameter changes, we observed progression into the formation of arterial-venous shunts (Fig. 11E-I). Although PBS-treated embryos survived, three out of five ephrinB2 treated animals died 8-10 hours after treatment. Given that ephrinB2 application on the yolk sac did not affect the survival of the embryos, we presume that they may die because of formation of the arterial-venous shunts.
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Discussion |
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Careful inspection of the morphological events underlying the separation of the arterial and venous system within the chick embryo yolk-sac plexus revealed a novel key event necessary for arterial-venous differentiation: the disconnection of small caliber sized vessels from the growing arterial tree starting at the most proximal part and progressing distally. Using detailed in vivo time-lapse observations of the initial arterial plexus starting at 15 ss for 24 hours, we observed that small caliber vessels connected to the stem of the forming vitelline artery are disconnected. On casts obtained from embryos at 21-35 ss, these disconnected vessels are visible as blind-ending sacs connected to the main branch. We subsequently observed that this process repeated itself towards the more distal parts of the vascular tree. The small vessels disconnected from the arterial system are subsequently reconnected to the venular plexus through specific projections, apparent as sprouting towards the developing venular plexus. After establishing connection with the primary venous system, they will start to carry flow again. To our knowledge, such a morphogenic event with potential relevance for arterial/venous growth has not been previously shown in vivo.
Our observations raise several important issues with respect to the
understanding of arterial-venous differentiation. First, we noted the
remarkable speed at which arterial-venous differentiation is achieved: only 12
hours elapse between the formation of the vitelline artery and the formation
of the vitelline vein. TUNEL staining failed to reveal significant cell death
in the yolk sac vessels during arterial-venous differentiation. Endothelial
divisions are uniform in the yolk sac and are thus unlikely to account for the
remarkably speed of the formation of the major vessels such as the posterior
vitelline vein (Fabian and Wilt,
1973). According to the current literature
(Risau, 1997
), several
possibilities for vessel remodeling have been described: sprouting,
intussuception, angioblast differentiation and migration, and pruning. DiI
injections and QH1 staining labeling EC membranes of developing yolk sac
vessels have not revealed a significant number of sprouts in yolk sac vessels,
while such sprouts were readily observed in intersomitic vessels. Sprouting
angiogenesis does therefore not seem to constitute the major operational mode
during yolk sac remodeling. Intussuception may better account for some aspects
of the process we describe here. Djonov has described intusseptive
arborization (IAR) of vessels in the chorioallantoic membrane
(Djonov et al., 2002
). During
IAR, a capillary plexus is transformed into large and small vessels by the
formation and alignment of tissue pillars alongside the future large vessels,
which are subsequently split off the capillary bed. This process may apply for
the formation of the most proximal part of the vitelline artery between 15 ss
and 22 ss. However, actual physical disconnection of small vessel segments has
not been described in IAR, or any other angiogenic remodeling process.
Disconnection differs from shear driven vessel regression as neither the main
branch nor the disconnected vessel diameters have significantly changed.
However, it can be explained based on physical principles. This form of
pruning is not a reduction of diameter as a function of shear inside the small
side branch, but direct breakage of the connection in the region of the
intersection of the main branch and the smaller caliber side branch, as a
function of flow in the larger vessel
(Fleury and Schwartz, 2001
).
The limited amount of apoptosis in yolk-sac vessels, together with the
observation that disconnected vessel are re-used to make veins, indeed
supports the idea that not vessel regression, but disconnection is the main
pruning mechanism, enabling use and re-use of vascular segments.
We have not yet addressed the possible role of angioblast migration in yolk sac remodeling. It remains possible that vessel disconnection or reconnection is achieved by the directed incorporation of migrating angioblasts into these sites. QH1 stainings have failed to detect isolated angioblasts at sites of vessel disconnection. Quail-chick chimera studies should allow to directly test if migrating angioblasts are incorporated into vessel breakage sites. Nevertheless, the observations we describe suggest that yolk sac vessels can be considered as `bricks' that are used and re-used by the growing vascular system according to its needs.
This view implies that yolk sac arteries and veins are plastic and
regulated by external cues, such as flow. To address this question, we altered
the perfusion pattern using physical approaches. Complete inhibition of flow
to the yolk sac was achieved in the no-flow model. No arterial-venous
differentiation and patterning was obtained in the absence of perfusion, in
spite of continued growth of the unpatterned yolk sac blood islands over a
considerable period of development (at least 7 days). A functional perfused
loop between heart and yolk sac vessels therefore appears necessary to
initiate formation of arteries and veins in the yolk sac. Examination of the
expression of ephrinB2 in no-flow yolk sacs showed that some areas of the
undifferentiated capillary bed expressed this marker, whereas others did not.
These data suggest that initiation of arterial marker expression is
independent of flow and perhaps genetically predetermined. Indeed, observation
of the normal expression pattern of ephrinB2 and NRP1 has already shown that
expression is initiated in the embryo prior to the onset of flow
(Herzog et al., 2001; Moyon et
al., 2001). Second, normal flow patterns in the yolk sac were altered in the
incision and ligation models. In the incision model, the vitelline artery on
the incised side did not develop; instead, a single vein formed in the area
normally occupied by the vitelline artery. In the ligation experiments, the
arterial bed on the ligated side was venularized. Moreover, growth of arteries
was induced in areas normally devoid of arteries such as the posterior region
of the embryo, normally occupied by the posterior vitelline vein and the
anterior region, normally occupied by the anterior vitelline vein. These
experiments indicate that flow determines both the arteriolar and venular
patterning. Pre-existing arterioles were incorporated into the newly
developing venular system, if they were oriented in the direction of the newly
induced venous flow. Conversely, pre-existing veins could be incorporated into
expanding arteries. Expression of mRNA encoding ephrinB2 or NRP1 was rapidly
downregulated after ligation of the vitelline artery. Already 10 minutes after
ligation, expression of NRP1 is reduced in the distal tips of the arterial
tree. After 1 hour, the loss of expression becomes more pronounced and after 4
hours it is completely lost. In the unligated side, normal expression patterns
were observed. Subsequently we investigated whether the venularized arterial
tree can express NRP2 and Tie2, genetic markers of venous endothelium.
Expression of both mRNAs was observed 12 hours after ligation, indicating that
EC in the new plexus acquired a venous identity. Interestingly, the kinetics
of induction of venous markers were slower than those of loss of arterial
markers. The manipulated vessels therefore seem to undergo a phase of loss of
arterio-venous identity.
As it may be argued that the loss of ephrinB2 and NRP1 expression is due to
absence of perfusion and subsequent death of the arteries, we examined in
close detail the flow changes acutely after ligation. We observed that after
ligation the arterial plexus is still perfused but with blood attracted from
the marginal vein; hence, blood of venous origin. To investigate the changes
in more detail, we also re-opened the ligation allowing reperfusion of the
right vitelline arterial tree with arterial blood. Re-opening the ligation and
allowing 4 hours of re-perfusion resulted in the re-emergence of NRP1
expression. This clearly shows that flow can control NRP1 and ephrinB2
expression in vivo. These data strongly indicate that the yolk sac vasculature
patterning is not genetically committed but shows plasticity in the sites of
arterial-venous differentiation, and subsequent patterning depending on the
local hemodynamic conditions, flow. Indeed, physical modeling data that
incorporate the time-lapse observations obtained in vivo show that flow can
explain and moreover is sufficient to explain arterial and venous patterning
(Fleury and Schwartz, 2000;
Fleury and Schwartz, 2001
).
This for the first time clearly shows that flow may be the master regulator of
the arterial-venous differentiation and patterning process.
Although the yolk sac remodeling may be plastic and regulated by flow,
several steps in the formation of the cardiovascular system may require solely
genetic predetermination control. We postulate that formation of a contracting
heart, the in- and outflow tract, the projection of arteries from the aorta
towards the target organs, the connection to draining veins and the inflow
tract, thus all steps needed to make a functionally perfused loop between
heart and developing organs, are genetically driven. These postulates are
confirmed by the expression pattern of genes specific for arterial
endothelium. Gridlock, NRP1 and Delta-C can be expressed in presumptive
arterial endothelium before the onset of flow
(Moyon et al., 2001a;
Smithers et al., 2000
;
Zhong et al., 2000
).
As the flow signal has to be transmitted into morphogenic events we
subsequently investigated the role of ephrinB2 and EphB4, molecules previously
implied to be crucial for arterial-venous differentiation and patterning. The
process of vessel disconnection and reconnection described here indeed shows
remarkable similarities with the function of these molecules as described in
the nervous system, repulsive and attractant signaling
(Kullander and Klein,
2002).
Expression of ephrinB2 in the main arterial branch, in conjunction with expression of EphB4 in the disconnected vessel theoretically could induce separation of arterial and venous endothelium. Such a mechanism would provide a theoretical explanation of the proposed models on ephrin/Eph interaction in the vascular system. To test whether ephrinB2/EphB4 may be involved in micro-events such as vessel disconnection/reconnection, or more macro events like the global patterning of arteries and veins, we applied recombinant ephrinB2 and EphB4, clustered and unclustered, on the chick embryo yolk sac. Neither application of recombinant ephrinB2 or EphB4 protein resulted in major changes in arterial and venous patterning. We were also not able to demonstrate a significant effect in the disconnection and reconnection process.
Although application of ephrinB2 and EphB4 on the yolk sac did not provoke
any overt morphological changes, application on more mature arteries and veins
in the E4 allantois induced the formation of large arterial-venous shunts.
EphrinB2 induced effects in the venous plexus: more side-branches were
detected. Subsequently the arterial plexus responded with huge diameter
increases of arterioles, finally progressing in the formation of
arterial-venous shunts. Vasodilation could not account for the huge
enlargement of the arteries, as previous studies already showed that allantois
vessels are always maximally dilated to allow adequate oxygen uptake
(Dusseau et al., 1986). These
diameter changes probably have a structural nature and have to be explored
further. In literature that addresses collateral formation in adult vascular
beds, the phenomenon of structural outward remodeling of the arteries is
referred to as arteriogenesis (Schaper and
Buschmann, 1999
). Interestingly, embryonic and adult
arteriogenesis are both perfusion driven. In fact, modeling studies have shown
that adaptation to flow induced shear stress alone, will result in the
formation of large arterial-venous shunts
(Hacking et al., 1996
). It is
therefore tempting to speculate that application of ephrinB2 enhanced shear
stress sensing, hence, enabling the formation of shunts. Given that ephrinB2
binds to veins, enhanced shear stress sensing would be mediated by a different
molecular mechanism.
At the molecular level, the in vivo effects of ephrinB2 suggest that there
is active forward signaling through the EphB4 receptor at this stage of
development. Application of EphB4 to the allantois also resulted in the
formation of arterial-venous shunts; however, these connections appeared
smaller in dimension in comparison to ephrinB2. Detailed investigation of the
kinetics of this process revealed more differences with respect to ephrinB2.
We observed that EphB4 enhanced the distal outgrowth of the arterial tree into
areas occupied by veins. The distal tips of the veins regressed, resulting in
a smaller venous tree. During subsequent stages, these regions are invaded by
arteries. Hence, EphB4 may be needed for arteries to invade presumptive venous
domains. This in vivo effect of EphB4 in E4 allantois suggests the presence of
reversed signaling through ephrinB2. These observations are compatible with
the phenotypes observed in mouse mutants for ephrinB2 and EphB4, which suggest
that reverse signaling through ephrinB2 is responsible for the observed
defects in arterial-venous differentiation
(Adams et al., 2001;
Gerety et al., 1999
;
Wang et al., 1998
). Further
confirmation for these results will await the generation of mutants deficient
in the kinase domain of EphB4. The allantois data suggest that ephrin/Eph
signaling at this stage of allantois development, is required for the
maintenance of this system, by inhibiting the mixing of arterial and venous
EC. Interestingly, application of ephrinB2 during the period E5-E10, failed to
produce similar effects, suggesting time-dependent changes in the efficacy of
this signal transduction pathway.
Such time-dependent changes could also account for the failure to detect
any overt morphological changes in the yolk sac after ephrinB2/EphB4
application. Collectively, our data suggest that ephrin application has
effects in allantois vessels and not in yolk sac vessels. A theoretical
explanation for the observed difference could be that ephrinB2 protein does
not reach the yolk sac vessels. We feel this unlikely, given that protein
binding is observed. It is furthermore possible that protein cannot elicit a
proper signal transduction cascade in the yolk sac, perhaps because a member
of the downstream cascade is absent. This occurs in other receptor/ligand
systems during embryonic development
(LeNoble et al., 2000). It may
also be that ephrin/Eph signaling in the yolk sac is already maximally active,
and the added proteins, which were previously described as agonists in both
neural and endothelial cells (Wang and
Anderson, 1997
; Füller et
al., 2003
) do not increase signal transduction. In this case,
application of ephrinB2 monomers or other antagonistic forms should provoke an
effect in the yolk sac. Future experiments will answer to these questions.
The observation of impaired yolk sac remodeling in the absence of flow or
perfusion may have important impact on the interpretation of yolk sac
remodeling events in mice deficient for genes implicated in vascular
development. For example, HiF1-/-,
VE-cadherin-/-, activin receptor-like kinase 1-/- and
connexin 45-/- mice all show severe impairment of yolk-sac
remodeling. However, these mice also exhibit cardiac defects or failures in
outflow tract remodeling (Carmeliet et al.,
1999
; Iyer et al.,
1998
; Krüger et al.,
2000
; Ryan et al.,
1998
; Urness et al.,
2000
). Perfusion in these mice is thus certainly perturbed leaving
open the possibility that part of the vascular phenotype observed in the
yolk-sac of these null mice is in fact due the absence of perfusion, and not
directly due to the targeted deletion of the gene. It has been reported that
the yolk sac of the original ephrinB2 knockout mouse is perfused
(Wang et al., 1998
) and makes
a primitive arterial plexus. Hence, a functional perfused loop between heart,
aorta and yolk-sac must have been present. However, as both the ephrinB2
knockout, and the conditional Tie2-Cre ephrinB2 knockout
(Gerety and Anderson, 2002
)
display cardiac malformations as well as malformations of intraembryonic
vessels, it cannot be ruled out that the failure to drive the outgrowth of
arteries is partly due to a defect in the perpetuation of cardiovascular
perfusion.
In conclusion, we show that arterial-venous differentiation is a flow driven highly dynamic process that exhibits a high degree of EC plasticity. Understanding the regulation of EC plasticity with respect to vessel identity has obvious important implications for the use of veins in coronary bypass surgery, restenoses and therapeutic arteriogenesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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