Fig. 4. Movement of individual cells of the AVE. (A,B) Tracks of eight cells overlaid on the last frame of the movie so as to show the paths they took to reach their final position (Fig. 2, Movies 2 and 3). Cells are labelled c1 to c8. Cells were tracked after 1 hour and 48 minutes had elapsed because they could not be reliably distinguished before this. Cell movements are rather direct during distal to proximal migration, but become highly convoluted once they start spreading laterally. Cells 4 and 7, and 5 and 6 are sister pairs, and hence share a common track prior to their division. (C) A quantitative measurement of cell behaviour was obtained by calculating, for each cell and at each time interval of 12 minutes, the ratio of its proximal displacement to its lateral displacement. The mean values for all eight cells were calculated for each time point, and are plotted against time in culture. Initially, the ratio tends to be substantially greater than unity, indicating that displacement is predominantly proximal. At approximately 5 hours (red arrow), when cells reach the boundary of epiblast and extra-embryonic ectoderm, the ratio decreases to less than one, indicating that motion is predominantly lateral. (D) Inspection of panels A and B suggests that cells move to the left on reaching the boundary between epiblast and extra-embryonic ectoderm. This is an artefact introduced by the fact that the embryo `rolled' slightly to the left during culture (see Movie 2, at 7 hours of culture). The effects of rolling can be abrogated by calculating the separation between cells, and D plots the separation between two representative pairs of cells, as well as the average distance moved by the four cells during each 12-minute time interval. Cells 1 and 3 move apart during culture, whereas 2 and 4 come closer together, indicating that cells do not behave in a coordinated manner on reaching the extra-embryonic ectoderm. Though the distance covered by cells in each time interval varies widely, it does not show any trend over the course of culture, indicating that the cells do not slow down or speed up. Scale bar: 50 µm.