Figure 5

Tracking H2B-GFP reporter expressing cells in the visceral endoderm of an E5.5 TCF/Lef:H2B-GFP embryo. Rendered images of 3D time-lapse data of E5.5 TCF/Lef:H2B-GFP embryo acquired on a spinning disc confocal (A-D), with high magnification detail in greyscale shown beneath (A'-D' and A'' = D''). Duration of time-lapse experiment was 9 hours 46 minutes and 21 seconds (t = 9:46:21). Individual cells identified by H2B-GFP nuclear-labeling were color-coded (open circles) and tracked using the spots function in Imaris (Bitplane, Inc.). First panel (A, t = 0) depicts the initial state, with lower panels depicting high magnification views of tracked cells and reference cells. At t = 3:48:02, (B) the first tracked cell division occurs and pushes the bottom-most reference cell to the left. Cell divisions are highlighted with a white outline on the color-coded open circles. In panel C, (t = 6:30:54) nearest-neighbor relationships are preserved subsequent to the near-synchronous division of five cells, despite substantial growth of the embryo. In the final panel, (D, t = 9:46:21) the non-dividing reference cells (yellow closed circles on lower series of panels) have shifted in their relative position and the distance between them has increased; however, the daughter cells produced by previous cell divisions along the left have maintained their nearest-neighbor relationships. The constriction in the range of angle between the cells, as well as the distance between reference cells, suggests that circumferential (lateral) expansion of the embryo is greater than the proximal-distal (longitudinal) growth. Scale bars: 30 μm, upper panel; 20 μm high magnification images, depicted in lower panels.