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Fig. 3. Epithelial cells show dynamic cell movements that differ in distinct regions of the gland. (A) Still frames from time-lapse supplementary material Movie 4 of E13 SMG epithelia. The frames are single confocal sections through the center of the gland with epithelial cells labeled by GFP (green) and fibronectin labeled with Alexa Fluor-647-FN (red). (B) Individual cell movements were manually tracked in 3D; an example bud track is shown in white overlaid on an XY projection with XZ (top) and XY (left) projections. (C) Representative tracks of migrating cells from the bud interior (black) or duct interior (green) compared to bud cells that had contacted the basement membrane (BM, purple) displayed as a 3D rose plot (X, Y, and Z axis) with the origin of each track at 0, 0, 0. Cell tracks were quantified for velocity (T/t), displacement (D), total distance traveled (T), and meandering index (D/T) and displayed as bar graphs: mean ± s.e.m. On average, cells in buds traveled at higher velocities and for longer distances than duct cells, and cells that contacted the basement membrane traveled even faster and further than cells in the interior of buds. Cells were tracked for 5 hours; n=44 (bud), 29 (duct) and 45 (BM), averaged from six experiments. The Wilcoxon signed-rank test was used to calculate statistical significance of differences, *P<0.001, **P<0.0001 compared with bud cells. (D) After time-lapse imaging, the epithelia were fixed and stained with anti-E-cadherin antibody: E-cadherin (blue), GFP (green), and FN (red). All GFP-labeled cells (green) were inside of the basement membrane demarcated by FN, and all expressed E-cadherin. Bars, 50 µm (A), 10 µm (D).
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