1 Department of Anatomy and Cell Biology, University of Kansas Medical Center,
3901 Rainbow Boulevard, Kansas City, KS 66160, USA
2 Department of Biological Physics, Eötvös University,
Pázmány sétány 1A, Budapest, 1117 Hungary
* Author for correspondence (e-mail: clittle{at}kumc.edu)
Accepted 9 March 2004
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SUMMARY |
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Key words: Vasculogenesis, Endothelial cells, vß3 integrin, Time-lapse, Computational biology, Quail
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Introduction |
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The importance of the endothelial cell has long been recognized and there is rich literature documenting a multitude of endothelial cell regulatory pathways and adhesive behaviors, both cell-cell and cell-extracellular matrix (ECM). Even more effort has been expended on drug discovery, with investigators pursuing agents that either promote or prevent tissue vascularization. Arguably, however, the least understood and most important question facing vascular developmental biologists and tissue engineers is what are the general principles guiding morphogenesis of a functionally patterned array of endothelial tubes?
Various hypotheses have been proposed to account for the cell dynamics of
vasculogenesis. One suggests that endothelial cells migrate to pre- and
well-defined positions following extracellular guidance cues or
chemoattractants (Ambler et al.,
2001; Cleaver and Krieg,
1998
; Poole and Coffin,
1989
). Based on in vitro studies, another hypothesis proposes that
angioblasts first segregate into randomly placed compact clusters and engage
the surrounding ECM fibers. As a result of traction forces, ECM bundles
develop, which in turn later route the motile primordial endothelial cells
between clusters (Drake et al.,
1997
; Vernon et al.,
1992
; Vernon et al.,
1995
). A mathematical model demonstrated that suitable cell
traction forces, without substantial cell migration, are capable of forming
polygonal patterns reminiscent of the primary vascular plexus
(Manoussaki et al., 1996
).
The above concepts stress the importance of cell-ECM interactions during
vascular pattern formation. Previous studies have established the importance
of the vß3 integrin during vessel formation, because in situ
inhibition of
vß3 resulted in abrogation of vascular morphogenesis
(Brooks et al., 1994a
;
Drake et al., 1995
).
Additionally, tumor and skin graft models showed
vß3 antagonists
promoted tumor regression and apoptosis of angiogenic vessels
(Brooks et al., 1994b
;
Brooks et al., 1995
).
Owing to recent improvements in digital microscopy, it is now possible to
address directly how blood vessels form de novo
(Czirok et al., 2002;
Rupp et al., 2003
). Scanning
time-lapse microscopy and statistical analyses of the recorded biological
motion results in a simultaneous tissue- and cellular-scale characterization
of primordial endothelial cell behavior. Here, we have examined how perturbing
vß3 cell-ECM interactions influenced vascular patterning dynamics.
We conclude that subtle, specific alterations in endothelial cell behavior are
manifested over time as a highly amplified malformation of the primary
vascular plexus.
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Materials and methods |
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Embryo microinjection
Non-treated embryos were microinjected with Cy3 (Amersham Pharmacia
Biotech, Piscataway, NJ) fluorochrome-conjugated QH1 antibody (Cy3-QH1)
(Developmental Studies Hybridoma Bank, University of Iowa, Ames, IA) to label
quail vascular endothelial cells. Two 25 nl Cy3-QH1 injections (0.58 ng/nl)
were introduced bilaterally per embryo into the interstitial space between the
endoderm and splanchnic mesoderm. On the basis of several hundred embryos,
there is no evidence that microinjection of PBS, human serum albumin,
hybridoma `G' or immunoglobins, per se, interfere with normal embryonic or
vascular development, as judged by QH1 staining
(Drake et al., 1995;
Drake et al., 1992
;
Drake et al., 2000
;
Drake and Little, 1991
;
Drake and Little, 1995
)
(P.A.R. and C.D.L., unpublished).
Control embryos were injected with a non-function blocking antibody to the
v integrin subunit (MAB1953Z, Chemicon International, Temecula, CA), in
addition to Cy3-QH1. MAB1953Z (1 ng/nl) was introduced in four 25 nl
microinjections (13 embryos), one 25 nl microinjection at 4 ng/nl (2 embryos)
or four 25 nl injections at 4 ng/nl (5 embryos).
Experimentally perturbed embryos were injected with LM609 (MAB1976Z, Chemicon International, Temecula, CA), in addition to Cy3-QH1. MAB1976Z (1 ng/nl) was introduced in four 25 nl microinjections, two per embryonic side, or one 25 nl microinjection at 4 ng/nl (16 embryos total).
Embryo culture
Embryos were cultured as described by Rupp et al.
(Rupp et al., 2003). Briefly,
injected embryos were placed ventral side up on suture beds within a modified
culture chamber containing 3.5 ml Leibovitz-L15 medium supplemented with 2 mM
L-glutamine, 10% chick serum and 1% penicillin-streptomycin (GibcoBRL, Grand
Island, NY). A second paper ring was placed on top of the embryos, empty wells
were filled with sterile deionized H2O, and the chamber was then
sealed shut and positioned on a microscope stage insert within an attached
incubator heated to
38°C.
Image acquisition and movie assembly
Images for time-lapse analysis were acquired on a Leica DMR upright scope
(Leica Microsystems, Wetzlar, Germany) with attached Ludl BioPrecision stage
(Ludl Electronic Products, Hawthorne, NY). All imaging was carried out using a
10x (0.25 NA) objective with a 20 mm working distance and a Photometrics
Quantix cooled CCD camera (Roper Scientific, Tucson, AZ). A complete
description of the software for image acquisition and movie assembly is
described elsewhere (Czirok et al.,
2002). A more complete description of the instrumentation can be
found elsewhere (Czirok et al.,
2002
; Rupp et al.,
2003
).
In each experiment, images are acquired for three embryos. For each individual embryo, a rectangular area is created from 2x3 slightly overlapping microscopic fields. Multiple (10) focal planes for each field are recorded at 20 µm intervals, first in DIC and immediately thereafter in epifluorescence modes. The acquisition of the `z-stack' pairs is accomplished within a short period of time (typically a minute) ensuring the correct spatial registration of the corresponding DIC/epifluorescence image pairs. The practical result of this technology is that every position of the embryonic disc lies within one of the focal planes; thus, no feature is lost or out of focus.
Quantification and compensation of vascular drift
Image alignment was carried out as described in detail previously
(Czirok et al., 2002). Briefly,
to align an image pair, a pixel-by-pixel comparison is performed for a large
number of possible relative offsets. The best alignment is selected as the
offset, which results in the least average pixel-by-pixel difference between
the shifted image copies.
Embryos change their shape and position considerably during development. To provide an internal frame of reference, DIC images were aligned in such a way that major embryonic features (intersomitic clefts and the notochord) remained stationary in the resulting image sequences. The same translations were implemented onto the epifluorescence image sequences as well, keeping the DIC/epifluorescence image pairs in register.
To quantify and compensate for vascular drift, an initial vascular pattern
was selected lateral to the 3rd-6th somites. The movement of the pattern was
determined by successive re-alignments performed on the previously
motion-compensated image sequences, resulting in a sequence of offset vectors
dxi, where i is the frame index. The cumulative
displacement (Xi) of the structure visible in frame
i, relative to the steady anatomical features, is then given simply
as
.
Moreover, drift compensation can be achieved by shifting the image on each
frame by -Xi (Czirok et
al., 2002
).
Cell tracking and velocity
Manual tracking, using software described previously, followed the
two-dimensional projections of various primordial endothelial cells (PECs) and
endothelial structures (Hegedus et al.,
2000). Briefly, this software allows tracking the point of
interest in x, y and z directions over time. When performed,
this procedure resulted in the positions xa(t) of
a certain object a at various time points t. The velocity,
va(t), was calculated as the net displacement of
the geometrical center during 1 hour long time intervals:
,
where
t=1 hour.
Average displacement
To describe the persistence of cell motility
(Stokes et al., 1991),
da(
), the average distance of migration during a time
period of length
was calculated for each cell a and a wide
range of
, as
.
For a given value of
, the average {...}t includes
each possible time point t for which both
xa(t+
) and
xa(t) exists. In a similar fashion, an entire
cell population can be characterized by
,
where averaging extends over each cell of the population in addition to
t. The functional form of d is very characteristic for the
underlying motion. In the case of a mathematical random walk, d grows
as a square root of
, while for a highly persistent, straight motion
d is proportional to
.
Statistical analysis
Wilcoxon tests with significance level of P<0.05 were used to
compare the sets of data. This test is insensitive to variations in sample
size and does not assume a particular analytical form of the distribution.
Statistical errors of the d() displacement curves were
calculated as
E2(
)={d2a(
)a-{da(
)}2a.
For a fixed
, the significance of difference between two
d(
) curves was established by comparing the sets of
corresponding da(
) values. Each of these is
characterizing distinct cells and therefore is assumed to be statistically
independent.
Online supplemental material
A number of movies can be found at
http://dev.biologists.org/supplemental,
each specific for one of the first six figures. In all movies, endothelial
cells are labeled with Cy3-QH1 and appear white unless otherwise noted. All
still images found in figures that have been taken from movies, are inverted
so that the endothelial cells and structures appear as black on a white
background. The normal vasculogenesis of a HH stage 8 quail embryo is shown in
Movie 1, with corresponding still images found in
Fig. 1. The second movie (Movie
2) contains four panels showing vasculogenesis in two normal and two
LM609-perturbed embryos. Each panel represents one 400 µm x 400 µm
area that was analyzed. The upper right and lower left movies are depicted
with still images in Figs 2 and
6, respectively. A progression
of untreated primordial endothelial cell trajectories, also shown in
Fig. 3, can be found in Movie
3. Cellular extensions and retractions, at high magnification, can be observed
in the three panels of Movie 4. The left panel shows the DIC image while the
middle panel displays the Cy3-QH1 pulse-labeled cells in black. The third
panel is a composite of the epifluorescence (red) and DIC images. The last
movie (Movie 5) displays how introduction of LM609 perturbs normal
vasculogenesis in a stage 8 quail embryo. For a more complete description,
including movie rates, please read the movie captions at
http://dev.biologists.org/supplemental.
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Results |
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The labeled extracellular QH1 epitopes were largely preserved during the
recorded time period (Fig. 1;
see Movie 1 at
http://dev.biologists.org/supplemental)
(see also Czirok et al., 2002).
A terminal QH1 immunolabeling at 12 hours, with a different fluorochrome
(Cy2), was performed after fixation of the recorded specimen allowing the
comparison of the final vascular structure with its pulse-labeled component.
As Fig. 1D demonstrates, the
pulse-labeled cells are randomly distributed in the final vascular structure.
The relatively low concentration of pulse-labeled cells demonstrates the
importance of new endothelial cell recruitment during incubation, most notably
in caudal regions.
The binding of QH1 antibodies to endothelial cell-surface epitopes does not cause any detectable perturbation. Indeed, the final vascular pattern exhibited by QH1-injected embryos is not distinguishable from the vascular patterns observed in control-injected embryos or embryos that develop in ovo. We have also performed labeling with QH1 Fab fragments such embryos are indistinguishable from embryos labeled with intact QH1 IgG except that there is a diminution in the fluorescence staining intensity (data not shown).
QH1 staining pattern
Images acquired during the first 3 hours of recording show the `clearing'
of QH1 antibody, manifested as the disappearance of diffuse background
fluorescence (Fig. 2; see Movie
2 at
http://dev.biologists.org/supplemental).
QH1 homogenously stains endothelial structures for the ensuing 3-4 hours,
after which time the immunofluorescence becomes punctate. Most QH1+
foci initially appear as fine granular structures that originate from much
larger patches of fluorescence. The median distance between neighboring foci
is 10 µm, and most pairs of adjacent foci move at least 25 µm apart
during the observation period.
Hierarchy of vasculogenic events
The primary vascular plexus is created from a combination of processes,
each operating on different length scales. Accordingly, we distinguish: (1)
tissue deformations that passively convect PECs as the embryonic plate folds
and elongates; (2) `vascular drift', an ordered medial movement of the entire
vasculature; (3) structural rearrangement of formed vascular polygons; (4) PEC
migration along existing endothelial cord structures; and (5) cellular
extensions/retractions across avascular zones that form or remove links within
the network. In the following we investigate each in more detail.
Tissue-scale processes
In early vertebrate embryos, vasculogenesis occurs contemporaneously with
major morphogenic processes that profoundly influence the vascular pattern.
This is most clearly visible at the anterior intestinal portal (AIP), where
its regression is closely coupled to endocardium formation (data not shown).
Here, we restrict our analysis to regions where such gross deformations do not
occur: 400x400 µm areas were selected lateral to somites 3-6 at a
distance of 160 µm from the embryonic axis (cyan colored boxes in Figs
1 and
5). These areas are represented
at higher temporal and spatial resolution in Figs
2 and
6, respectively. These regions
are distant from both the AIP and Hensen's node, sites of profound tissue
deformations, and exhibit representative endothelial cell motility including
vascular drift and cellular protrusive activity.
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Dynamics of cell assemblies
The tissue-scale movements described above are usually associated with
structural rearrangements in the vascular network. The most frequent
rearrangement is vascular fusion, whereby distinct endothelial tubes fuse to
form a common sinus. This process is clearly shown in Figs
1 and
5, where the vascular
structures marked by green circles approach each other and coalesce to form a
nascent sinus venosus (see Movie 5 at
http://dev.biologists.org/supplemental).
Vascular fusion also occurs during the formation of the dorsal aortae.
Cell migration
Local trajectories of individual PECs can be established relative to the
surrounding vascular network by subtracting vascular drift
(Fig. 3). Each fluorescent foci
can be traced back either to a cluster of endothelial cells or to an avascular
area, where PECs appear de novo. These newly appearing cells move quickly
until their incorporation into an existing vascular structure (PEC #1,
Fig. 3). Once part of a
vascular cord, PEC speed is usually reduced, and when moving, PECs remain in
close vicinity of other endothelial cells.
There is a significant variation of motile activity within the endothelial cell population, as the varying trajectory behaviors demonstrate (Fig. 3; see Movie 3 at http://dev.biologists.org/supplemental). The median PEC velocity is 5 µm/hour, but a few cells move with speeds up to 40 µm/hour. Most PECs move on the vascular network from a lateral position to a more medial one.
Cell protrusions
Cells, exploring their environment, form extensions or `sprout' into
avascular zones. These protrusions can contact other extensions or endothelial
cords, thereby creating new connections and vertices in the primary vascular
pattern (Fig. 2, blue
arrowheads). Conversely, existing cell-cell connections are also observed to
retract (Fig. 2, red
arrowheads). The frequency of protrusion formation is observed to diminish
over time.
Higher temporal and spatial resolution reveals the creation of new connections between two adjacent endothelial cell clusters (Fig. 4; see Movie 4 at http://dev.biologists.org/supplemental). Cell protrusions can extend at a rate of 2 µm/minute into the surrounding avascular area, where they can bend, further elongate or retract. A stabilized protrusion may later be reinforced by subsequent addition of cells. No correlation between the direction of protrusive activity and the position of neighboring cell clusters was recognized, suggesting a random directionality of protrusions.
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Qualitative characterization of vascular dynamics revealed a substantial effect in the LM609-injected embryos (see Table 1). To allow a quantitative assay of LM609-induced changes, embryos injected at the five-somite stage were selected for further analysis. Introduction of LM609 was found to substantially reduce vascular drift (Figs 5 and 7). The cumulative displacement of an unperturbed vascular pattern can typically approach 80 µm within a 10-hour time period with the intensity of this movement diminishing with time. The presence of LM609 strongly reduces these displacements, especially early on (88% at 4-hour time point). As a consequence, fewer PECs arrive at medial positions, thus disrupting vascular fusion at the dorsal aortae, while fusion clearly continues at the more cranial sinus venosus.
|
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(1) To characterize the ratio of the slow versus fast moving cells within
the population, the velocity distribution function was determined
(Fig. 8). The average motility
in LM609 treated embryos exhibited a significant, 30% reduction
(P<0.05). The shape of the distribution, however, remained similar
to an exponential distribution, indicating a continuous spectrum of cell
activity (Czirok et al., 1998;
Upadhyaya et al., 2001
).
|
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In summary, normal endothelial cells exhibit local protrusive activity,
motility along pre-existing polygons, engage in structural rearrangements, and
have a lateral-to-medial drift similar in nature to sheet-like migration.
Addition of an vß3 integrin inhibiting antibody most dramatically
reduces endothelial cell protrusive activity and medial vascular drift. By
contrast, microinjection of a non-function blocking antibody to the
v
integrin subunit had no observable effect on vascular drift, PEC movement or
protrusive activity at concentrations ranging from 100 to 400 ng. Similarly,
in limited trials, injection of a function blocking
vß5 integrin
antibody appeared to have no effect on endothelial cell dynamics at the stages
studied (P.A.R., A.C. and C.D.L., unpublished).
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Discussion |
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Based on an exhaustive analysis of QH1 immunostaining patterns, Coffin and
Poole (Coffin and Poole, 1988) proposed a medial migration of angioblasts
destined to form the dorsal aortae. More recent work in zebrafish and
Xenopus, also demonstrated medial migration of angioblasts, arising
in the lateral plate mesoderm, contributing to the formation of the axial
vessels (Cleaver and Krieg,
1998; Fouquet et al.,
1997
). In addition, chimera studies have confirmed the migratory
potential of PECs. In a number of avian experiments, quail-derived graft cells
were found to disperse as far as 400 µm
(Klessinger and Christ, 1996
),
in cranial, caudal, lateral and medial directions within their host
environment (Noden, 1989
;
Noden, 1990
;
Pardanaud and Dieterlen-Lievre,
1999
; Poole and Coffin,
1989
; Wilting et al.,
1995
; Wilting and Christ,
1996
). Similar results were obtained with mouse-quail chimeras
(Ambler et al., 2001
). These
studies demonstrate that grafted angioblasts are unrestrained, invasive cells
capable of motility as individuals or small clusters. However, grafting
experiments have the intrinsic difficulty in that subpopulations of xenograft
cells may not necessarily reflect endogenous behavior, in situ. Ambler and
colleagues addressed this by labeling presumptive endothelial cells by DiI
injection. As was observed in chimera studies, labeled angioblasts migrated
significant distances (Ambler et al.,
2001
).
Observations of in vivo cell motility are becoming more common with
advances in imaging technology. GFP-constructs have been used in zebrafish
studies to visualize the later stage vascularization behavior of angiogenesis
(Lawson and Weinstein, 2002).
Unfortunately, direct motility during zebrafish vasculogenesis was not
reported and, moreover, early fish vascular morphogenesis seems to be distinct
from that of warm-blooded vertebrates
(Weinstein, 1999
).
Capitalizing on the quail-specific endothelial cell surface marker, QH1, we
mapped cell movements during primary quail vasculogenesis over an area
encompassing virtually all of the area pellucida. In addition to sampling a
wide area, our technique permits the tracking of a large population of tagged
cells and the quantitative analysis of digital image files. As the size of a
typical endothelial cell is in the range of 10-20 µm, it is reasonable to
assume that an approximate one-to-one relationship exists between labeled
cells and QH1+-foci. Thus, QH1+-foci serve as useful
indicators of PEC position, especially if their displacements are much larger
than an average endothelial cell.
Our time-lapse data showed global medial drift of the entire vascular
ensemble a finding that directly confirms the conclusions of Coffin
and Poole (Coffin and Poole, 1988). However, the extent of this motion was
much larger than anticipated: not only did cells forming the dorsal aortae
migrate, but the entire primordial vascular plexus translocated medially as
well. The highly ordered nature of this motion was also unexpected
instead of resembling trajectories of independently migrating cells or even
groups of cells, such as occurs with neural crest migration, the process more
closely resembles that of cell sheet-migration, albeit a sheet containing
`void' areas. Interestingly, extracellular matrix displacement mappings also
reveal similar global medial displacement tendencies
(Czirok et al., 2004),
presumably reflecting the global tissue deformations characteristic of
gastrulation and neurulation. The finding that LM609 substantially reduces
medial drift, strongly suggests that endothelium-ECM interactions are required
to produce this motion. It is possible that the composite vascular network is
a single structure moving within or on the ECM. Alternatively, the vascular
network may be embedded within and moving in concert with other cell groups
migrating medially.
We documented the motile activity of individual PECs superimposed upon tissue deformations and vascular drift. In most cases, the cells move along tracts of existing vasculature, but a few PECs sprout into avascular areas as well. The trajectory of individual motile cells is highly persistent with little frequency of direction reversal. As PECs, in the immediate vicinity, can be observed to migrate in opposite directions, this directed motility is more likely to be a cell autonomous property rather than the consequence of an external directional guidance system (i.e. no `local' chemotactic gradient). The two types of endothelial cell motility (drift versus individual) differ mainly with respect to their substrates: while tissue-scale movement and protrusive activity is highly integrin-dependent and requires an active engagement of the ECM, PEC motility along existing vascular structures appears to rely more upon cell-cell interactions.
The LM609-induced reduction in PEC motility is reflected by the persistence of small endothelial structures and cell clusters. A cell that is associated with a small endothelial segment can migrate freely upon that structure until it attempts to venture into an avascular zone, which, in an LM609-treated environment, is largely prohibited. As LM609-perturbed cells still show persistent motion, the displacement of that cell is limited to the length of the endothelial structure to which it is confined, thus reducing both the distance migrated and the velocity of the cell.
Many investigators have shown that blocking functional vß3
integrins reduces vascularization. Our results now directly connect this
morphological finding with the reduced motility of endothelial cells and the
vascular network exposed to LM609, whether it is by physically inhibiting
interactions with the matrix, altering signaling pathways or a combination of
the two. Our experimental data regarding the role of
vß3 integrins
in endothelial cell dynamics seem to contradict observations obtained by mouse
knock-out models (Bader et al.,
1998
; Hodivala-Dilke et al.,
1999
). The most likely explanation of this apparent contradiction
is that PECs have redundant mechanisms by which they can engage the ECM and
are able to exert feedback control over the expression of various integrin
receptors. Therefore, fully expressed, but blocked, receptors yield remarkably
different cell behaviors than a genetically missing protein.
The dynamic cell motility data allows one to refine hypotheses regarding
PEC behavior during vasculogenesis. First, the motile activity and
directionality of endothelial cells is strongly influenced by cell-cell
interactions. Second, protrusive activity or sprouting appears to be the key
mechanism used to generate new vascular cords. Third, only a small fraction of
PECs exhibits this latter type of protrusive behavior. Finally, to our
surprise, we failed to find any correlation between the directionality of the
protrusive behavior and the position of the surrounding QH1+ cell
clusters. In fact, our data are consistent with protrusion formation occurring
in a random fashion with no preferred direction. The detection of neighboring
endothelial structures by motile endothelial cells seems to involve extended
filopodia as proposed by Flamme and colleagues
(Flamme et al., 1993). This
type of protrusive behavior is reminiscent of `angiogenic sprouting', a
process thought to be characteristic of later vascular development. Thus, the
differences between angiogenesis and vasculogenesis seem to be further blurred
not only can de novo formed PECs become incorporated into later stage
angiogenic sprouts, but sprouting or protrusive activity also seems to be a
fundamental process needed to generate a primary vascular plexus.
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ACKNOWLEDGMENTS |
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Footnotes |
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