Figure 4: ECM expansion and cell volume enlargement account for cell spreading.

(a,b) Col2a antibody staining (red) of frozen sections from the chick metacarpal expressing cytoplasmic-GFP (green). (a) Low magnification view. (b) Enlarged image of the region in the white box in (a). Inset of (b) shows sample counterstained with DAPI (blue) to reveal the locations of cell nuclei (three experiments; n=4 per experiment). (c,d) Cell segmentation for voxel analysis. (c) Maximum intensity projections of five time points in the 4D live imaging of a chick metacarpal expressing GFP; the region enclosed within the expanding white box selected for voxel analysis, based on the same four cells on the boundaries (red dots). (d) Corresponding binary images provided clear identification of voxels as either ECM (black) and cell (white) volume. (e,f) Voxel analysis. (e) Total count of the number of dark and white voxels, denoting the volume occupied by ECM and cells, respectively, shows the expansion of both ECM and cell volume. (f) Decomposition of the increase in ECM and cell length along the x and y axes, expressed as the percentage of the length at t=0 (100% denotes no change). (g,h) Results of computer simulations of cell trajectories, based on the model of tissue growth described in text and depicted schematically in Fig. 5. (g) Overlapping simulated and experimental (tracked) cell trajectories along the y axis depicted for six randomly chosen cells. Heat map in the insert depicts the errors of all simulated trajectories as a percentage of the experimental values showing that the absolute errors are always below 3%. (h) Total cell displacement length (t=55 h) along the y axis of all simulated cells are plotted against their initial y positions, displaying similar distribution pattern to the experimental ones; n=109 cells in (c–e). Scale bars, (a,b) 15 μm, (c,d) 50 μm.