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First published online 19 May 2004
doi: 10.1242/dev.01160

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vß3 integrin-dependent endothelial cell dynamics in vivo

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

View larger version (175K): [in a new window]   Fig. 1. Stages of early vasculogenesis. (A-C) Taken from Movie 1 at http://dev.biologists.org/supplemental. Changes in the Cy3-QH1 pulse labeled primordial endothelial cell population over a 12 hour time period, with corresponding DIC images (insets). (D) The merged image of the pulse-labeled PECs (red) and the post-experimentally Cy2-QH1 labeled vasculature (green). The red and green open circles mark the same PECs and endothelial structures whose movements are followed in each panel. Note the extensive lateral-to-medial movement of those foci lying most distal to the midline axis (red lines). By comparison, those in a more proximal position show lesser displacements. The cyan colored box indicates the field that is enlarged in Fig. 2 and serves as a frame of reference between panels. The box is 400x400 µm with the upper right corner centered on the lateral edge of the third formed somite (yellow asterisks, inset), 160 µm from the midline. All images are from the ventral aspect. The yellow arrowheads in C (inset) indicate the developing pronephros. sv, sinus venosus; nf, neural folds; ao, area opaca; nc, notochord.

View larger version (146K): [in a new window]   Fig. 2. Cell dynamics establishing the primary vascular plexus. Time points 1-11 show the cell configurations, at hour intervals, within the area delineated by the cyan colored box in Fig. 1. Each panel represents a 400x400 µm area enlarged from individual frames in Movie 1, and are shown in Movie 2 (see http://dev.biologists.org/supplemental). As a reference, the upper right-hand corner is centered on the lateral edge of the third somite pair 160 µm from the midline. The final panel is a merged image composed of the Cy3-QH1, experimental PEC label (red) and the post-experimental labeling of the vasculature using Cy2-QH1 (green). The open circles enclose the same endothelial structures on each panel to ease following movements. Note the lateral-to-medial movement of all denoted PECs. Green circles represent new features entering the field of view from a lateral position. Blue arrowheads indicate new cellular extensions and connections with existing polygonal structures. The red arrowheads represent retractions of vascular cords. In the last panel, a red arrowhead indicates where one connection once existed between two of the circled areas. In addition, some vasculature is formed (green) that is not recorded (red/yellow).

View larger version (146K): [in a new window]   Fig. 6. Vascular cell dynamics are vß3 integrin dependent. Time points 1-11, enlarged from Movie 5 and displayed in the lower panels of Movie 2, show the cell configurations (at hour intervals) within the area delineated by the cyan box in Fig. 5, and thus are directly comparable in location and stages with Fig. 2. Note the minimal movement and the lack of protrusions of LM609 treated cells. The annotations are the same as in Fig. 2.

View larger version (115K): [in a new window]   Fig. 3. Trajectories of individual primordial endothelial cells, relative to the surrounding vascular structures. Each panel is a 200x200 µm area with the embryonic midline on the right, encompassing a 10 hour period of normal development (see Movie 3 at http://dev.biologists.org/supplemental). Circles highlighted by yellow indicate the current position of representative PECs; lines show their trajectory up to the corresponding time point. Red and green are used to distinguish medially and laterally oriented motion, respectively. Even with the subtraction of the medial vascular drift, there is a heavily favored lateral-to-medial cell migration. In the final panel (12 hours), the experimentally labeled PECs (black) and cell trajectories (yellow or white) overlay the post-experimentally labeled vasculature (green). PEC #1 first appears in an avascular zone and quickly migrates to a chord structure.

View larger version (172K): [in a new window]   Fig. 5. Blocking vß3 integrin activity disrupts early vasculogenesis. (A-C) Taken from Movie 5 (see http://dev.biologists.org/supplemental), show changes in endothelial structures over a 10 hour period within an embryo injected with LM609, an vß3 blocking antibody. (D) The merged image of the Cy3-QH1, pulse labeled (red) and the post-experimentally labeled vasculature (green). In D, the disruption of the dorsal aortae and the polygonal network is clearly visible (see Fig. 1 as a normal reference). Clusters of vascular endothelial cells, characteristic for earlier stages (A in Figs 1 and 5) persist, lacking protrusions and connections. The red and green open circles indicate the same endothelial structures on each panel. By comparing with Fig. 1, a substantial reduction in the lateral-to-medial vascular drift is observed (red bars). The vasculature marked by green open circles is fused while being drawn into the developing endocardium. This motion seems to be unaffected by LM609 exposure. The cyan box delineates the field that is enlarged in Fig. 6 and serves as a frame of reference between panels. The position of this area as well as the markers, with the exception of one (is, injection site), is the same as in Fig. 1.

View larger version (130K): [in a new window]   Fig. 4. High-resolution dynamics of protrusive activity. Movie 4 (see http://dev.biologists.org/supplemental) shows the extension and retraction of cellular protrusions at high magnification over a 104 minute time interval beginning 2 hours and 10 minutes into the recording. (A) Three time points from Movie 4. The left column shows QH1 experimentally labeled PECs, the middle column depicts the corresponding DIC images and the right column is a composite of the two. Red and yellow asterisks denote the relatively fixed position of two neighboring endothelial cell clusters – the two asterisks do not move substantially during the 26 minutes. By contrast, the corresponding red and yellow arrowheads indicate the presumed tips of two cell protrusions – note that these protrusions converge over the 26 minute interval. Their contact at 182 minutes is marked by a red outline on the image denoted by a black asterisk. The corresponding composite image, on the right, demonstrates that the DIC feature outlined in red is labeled with the QH1 antibody. (B) The post-experimental labeling of the same field at 12 hours and 7 minutes (727'). The left image is the experimental QH1 labeled cells, while the middle frame displays the post-experimental QH1 vasculature. After 12 hours, the original features in A (asterisks) have moved slightly, but are traceable to the positions of the red circles (see Movie 4). The green arrowhead in the middle frame points out the continuity of contact between the endothelial cell clusters. The frame on the right shows the combined fluorescence from QH1 injected 12 hours earlier in red (or yellow), and the post-fixation QH1 fluorescence labeling in green (or yellow), yellow indicates positions of overlap. Scale bar: 225 µm. The time of image acquisition is shown in the lower corner of each epifluorescence frame.

View larger version (24K): [in a new window]   Fig. 7. Time dependence of vascular drift. Within the defined areas (cyan color boxes in Figs 1 and 5) the displacement of the entire vascular structure was determined using a frame-by-frame comparison of the fluorescence pattern. To eliminate adaptation artifacts occurring in the first hour of culture, displacements were measured from the second hour of recording. Data obtained from three normal (QH1 only), three anti-v (QH1 plus anti-v) and three LM609-injected (QH1 plus LM609) embryos are shown, each represented by a separate curve. Averages of control (normal and anti-v; blue) and experimentally treated (LM609; red) embryos are shown by the unbroken lines. Positive displacement represents movement in the medial direction. Exposure to LM609 resulted in a significant 88%, 73% and 70% reduction in endothelial structure displacement at 4, 6 and 8 hour time points, respectively.

View larger version (26K): [in a new window]   Fig. 8. Velocity distribution of primordial vascular endothelial cells. After subtraction of the vascular drift, trajectories of individual PECs were established (Fig. 3). Velocities were calculated as one hour displacements along these trajectories. The velocity distribution of normal (QH1 only), anti-v (QH1 plus anti-v) and LM609-perturbed (QH1 plus LM609) PECs is shown. The LM609-perturbed velocities of the PECs show a subtle increase in the slowest moving (0-5 µm/hour) population. The average motility of the population was reduced by 30%.

View larger version (34K): [in a new window]   Fig. 9. Average displacements, d, of normal and LM609-perturbed primordial endothelial cells as a function of the length of time interval, . Each curve is calculated from the trajectories of a least 40 PECs within a lateral area from each of three normal (QH1 only), three anti-v (QH1 plus anti-v) and three LM609-injected embryos (QH1 plus LM609). The form of the d() curve is highly characteristic for the underlying trajectories. As the plot with logarithmic scales (inset) demonstrates, the cells motility is superdiffusive and persistent (slope larger than 0.5). Blocking of the vß3 integrins results in a 40% reduction in the average displacements.