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Supplementary Movie Legends Page 1 Kamei et al., MS# 2006-01-00604A SUPPLEMENTAL MOVIE LEGENDS Supplemental Movie 1 This movie shows transmitted light time-lapse images of several human EC in a 3D collagen gel forming numerous intracellular vacuoles, some of which combine to form highly enlarged spaces within and between the cells. The time-lapse covers a period of about 11 hours, with approximately 60 minutes elapsed time per second of movie (QuickTime; 2.6 MB). Selected frames from this movie are shown in Figure 2a. Supplemental Movie 2 This movie shows transmitted light time-lapse images of a single human EC in a 3D collagen gel forming a greatly enlarged intracellular ”lumenal” space encompassing the vast majority of the inside of the cell. The time-lapse covers a period of about 24 hours, with approximately 100 minutes elapsed time per second of movie (QuickTime; 2.7 MB). Selected frames from this movie are shown in Figure 2b. Supplemental Movie 3 This movie shows two-photon time-lapse images of a ventral intersegmental endothelial cell in the trunk of a Tg(fli1:EGFP-cdc42wt)y48 transgenic zebrafish. Vacuoles rapidly appear, rearrange, and disappear within the cell. The time-lapse covers a period of about 4-1/2 hours, with approximately 15 minutes elapsed time per second of movie (QuickTime; 4.3 MB). Each movie frame is a reconstruction from a Z-series of 15-18 planes (with 2 µm between planes) collected at that time point. Selected frames from this movie are shown in Figure 2c. Supplementary Movie Legends Page 2 Kamei et al., MS# 2006-01-00604A Supplemental Movie 4 This movie shows two-photon time-lapse images of a ventral intersegmental endothelial cell in the trunk of a Tg(fli1:EGFP-cdc42wt)y48 transgenic zebrafish. A single vacuole within the cell enlarges and expands to become an enlarged lumenal space, eventually forming a ventral connection to the lumen of the dorsal aorta below. The time-lapse covers a period of about 3 hours, with approximately 30 minutes elapsed time per second of movie (QuickTime; 4.2 MB). Each movie frame is a reconstruction from a Z-series of 15-18 planes (with 2 µm between planes) collected at that time point. Selected frames from this movie are shown in Figure 2d. Supplemental Movie 5 This movie shows two-photon time-lapse images of the ventral portion of a developing intersegmental vessel in the trunk of a Tg(fli1:EGFP-cdc42wt)y48 transgenic zebrafish. Vacuoles appear within the cell and merge and coalesce to form a gradually enlarging lumenal space. The time-lapse covers a period of about 11-1/2 hours, with approximately 30 minutes elapsed time per second of movie (QuickTime; 8.9 MB). Each movie frame is a reconstruction from a Z-series of 15-20 planes (with 2 µm between planes) collected at that time point. Selected frames from this movie are shown in Figure 2e. Supplemental Movie 6 This movie shows two-photon time-lapse images of the ventral portion of a developing intersegmental vessel in the trunk of a Tg(fli1:EGFP-cdc42wt)y48 transgenic zebrafish. Vacuoles appear within the cell and merge and coalesce to form a gradually enlarging lumenal space. The time-lapse covers a period of about 11.5 hours, with approximately Supplementary Movie Legends Page 3 Kamei et al., MS# 2006-01-00604A 30 minutes elapsed time per second of movie (QuickTime; 8.9 MB). Each movie frame is a reconstruction from a Z-series of 17-23 planes (with 2 µm between planes) collected at that time point. Supplemental Movie 7 This movie shows transmitted light time-lapse images of two initially separate human EC in a 3D collagen gel. The two cells form intracellular vacuoles that combine to form a single large large lumenal space bounded by both cells. The time-lapse covers a period of about 19 hours, with approximately 100 minutes elapsed time per second of movie (QuickTime; 1.7 MB). Selected frames from this movie are shown in Figure 3a. Supplemental Movie 8 This movie shows transmitted light time-lapse images of a group of human EC in a 3D collagen gel. The cells sequentially incorporate into a multicellular group of cells bounding a single enlarging lumenal space. The accompanying movie 9 shows that the formation of this structure is accomplished without mixing of the cytoplasmic contents of the aggregating cells. The time-lapse covers a period of about 40 hours, with approximately 120 minutes elapsed time per second of movie (QuickTime; 9.4 MB). Each movie frame is a reconstruction from a Z-series of 4 planes (with 5 µm between planes) collected at that time point. Selected frames from this movie are shown in Figure 3c (bottom). Supplemental Movie 9 This movie shows composite red/green epifluorescence time-lapse images of a mixed group of differentially labeled human EC in a 3D collagen gel. The cells sequentially Supplementary Movie Legends Page 4 Kamei et al., MS# 2006-01-00604A incorporate into a multicellular group of cells bounding a single enlarging lumenal space. The accompanying movie 8 shows the corresponding transmitted light images of the same field of cells. Note that the frames in movie 9 only correspond to every sixth frame/time point in movie 8. The time-lapse covers a period of about 40 hours, with approximately 120 minutes elapsed time per second of movie (QuickTime; 7.9 MB). Each movie frame is a reconstruction from a Z-series of 4 planes (with 5 µm between planes) collected at that time point. Selected frames from this movie are shown in Figure 3c (top). Supplemental Movie 10 This movie shows a composite red/green time-lapse two-photon images of the ventral portion of a developing intersegmental vessel in the trunk of a Tg(fli1:EGFP-cdc42wt)y48 transgenic zebrafish injected intravascularly with 605 nm quantum dots. 960 nm twophoton excitation permits simultaneous activation of EGFP (green) and quantum dot (red) emission, which are then collected by separate PMT detectors. The movie shows successive labeling of preformed vacuolar compartments in the ventral-most endothelial cell and then the next more distal endothelial cell via transfer of quantum dots that are initially present only in the dorsal aorta. The time-lapse covers a period of about 2 hours, with approximately 10 minutes elapsed time per second of movie (QuickTime; 7.5 MB). Each movie frame is a reconstruction from a Z-series of 15 planes (with 2 µm between planes) collected at that time point.
