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. 2008 Dec 1;22(23):3268–3281. doi: 10.1101/gad.1725808

Figure 5.

Figure 5.

Pioneer and follower behavior. (A) Schematic representation of coculture experiments to investigate pioneer and follower behavior (green and orange colored cells used for illustration). Cell marking was based on one population stained with CellTracker (InVitrogen) and both populations stained with Hoescht. (B) FGFR1+ cells induce polarized movement in neighboring FGFR1− cells. FGFR1+ or FGFR1− cells were cocultured 1:1 with FGFR1+ or FGFR1− cells (four combinations). Cells in contact with the sheet margin in one population were tracked and their orientation measured (tracked cells listed first and neighbors listed in parentheses). A random orientation is 0.25. (C, left panel) Example of an image depicting pioneer (red) versus nonpioneer (green) locations at the sheet margin (defined as more versus less than half of the circumference in contact with the open space). White cells depict internal sheet cells. Cell boundaries were based on nuclear and F-actin stains. (Right panel) Pioneer cells are often FGFR1+ and are rich in actin ruffles. Picture is a close-up of the white box shown in the left panel with phalloidin staining shown in red, Hoescht staining shown in blue, and FGFR1+ cells shown in green. Lamellipodial ruffling is highlight with white arrows. (D) FGFR+ cells are enriched in pioneer positions. Bar graph shows the ratio of FGFR1+/FGFR1− cells in pioneer, edge and sheet positions. Plating ratio was 0.2 between FGFR1+:FGFR1− cells. (E) Diagram illustrating the positions from which directed motility measurements were taken for the “follower” experiments in F and G. (F) Follower behavior is lost inside sheets lacking VE-cadherin. Directed motility of cells was measured 150 μm from the sheet margin under various coculture conditions. Unmixed experiments represent homogenous cultures receiving the indicated siRNA treatment. Mixed population experiments show the directed motility for each population within a cocultured experiment (plated at a 1:1 ratio) (G) Directed motility for cells positioned at the sheet margin in monolayers treated with combined gene and/or control knockdowns.