Extended Data Fig. 7: Determination of total cell count and effective cell diameter.

a: Visualisation of the cell masks obtained by 3D segmentation⁵². (Left:) Slice of a confocal image z-stack of a 120 h old gastruloid, seeded from \({\overline{N}}_{0}=100\) cells, stained for phalloidin (orange) and DAPI (blue). (Right:) Phalloidin channel from left in grayscale overlaid with cell masks obtained by 3D segmentation (see Methods). Scalebar is 50 μm. b: Estimation of the discrepancy between 3D and 2D volume reconstruction. The pipeline presented in Extended Data Fig. 1c overestimates gastruloid volumes; we estimate by how much using the volume determined by 3D segmentation as a ground truth. Distribution of the error Err on the volume determined by 2D volume reconstruction \({V}_{2D}\),before 120 h and at 120 h, overlaid by a Gaussian distribution fit for each distribution. Vertical dashed lines correspond to the mean of each distribution. The ground truth 3D volume \({V}_{3D}\) was obtained from the 3D segmentation. Before 120 h, \({Err}=3.2\pm 8.2 \%\) (\(n=56\)). After 120 h, \({Err}=20.0\pm 11.2 \%\) (\(n=40\)). The volume was overestimated in both time classes but more so when the gastruloid elongated. Note that this evaluation of the discrepancy between 3D and 2D volume reconstruction is independent of the shrinkage factor (Extended Data Fig. 6) because 3D and 2D volume reconstructions are applied to the same shrunken gastruloid mounted with the phalloidin staining protocol. c: Scatter plot of the measured volume from 2D reconstruction \(V\) (corrected for the error determined in b) versus the total cell count \(N\) obtained by chemical dissociation (with the protocol in Extended Data Fig. 1e), for 492 individual gastruloids at different time points (color code) and with varying \({\overline{N}}_{0}\) (symbol). From \(V\) and \(N\) for each individually dissociated gastruloid an effective cell volume \({V}_{c}=V/N\) was computed, and from there we obtain the slope (black lines). The mean \({\bar{V}}_{c}\) for gastruloids aged from 24 to 48 h (before Chi-pulse) and the mean \({\bar{V}}_{c}\) for gastruloids aged from 72 to 120 h (after Chi-pulse) correspond to dashed and full lines, respectively. Inset shows correlation (\(r=0.78\)) of variability for \(V\) and \(N\) for sets of gastruloids with identical age and \({\overline{N}}_{0}\). The effective cell diameter \({d}_{c}\) can be obtained from the distribution of \({V}_{c}\), or directly from the slopes (see Methods and d). d: Distribution of the effective cell diameters \({d}_{c}\) per dissociated gastruloid, calculated from each effective single cell volume (\(V/N\)), before (red) and after (blue) Chi-pulse. Black lines are a Gaussian fit for each distribution. Vertical dashed lines correspond to the mean of each distribution. Before Chi-pulse, \({d}_{c}=16.0\pm 0.6\) μm (4.0%, \(n=206\)); after Chi-pulse, \({d}_{c}=13.9\pm 0.5\) μm (3.8%, \(n=286\)). This is evidence of a Chi-pulse-induced reduction in gastruloids’ effective cell size by ∼13% (linear dimension). e: Single cell volume distributions serve to reject noisy masks from 3D segmentation results. After an initial rejection of any 3D masks smaller than 10⁴ voxels, a bimodal distribution of the logarithm of single cell volumes \({V}_{c}\) (obtained by 3D segmentation of a 120 h old gastruloid with \({\overline{N}}_{0}=100\)) is fit by a two-component Gaussian mixture model (left). The mode in black corresponds to the distribution of small noisy masks, the mode in red corresponds to the distribution of well-segmented cells. Morphological closing is performed on the latter and the corresponding distribution of single cell volumes \({V}_{c}\) is shown in right panel, with noisy masks (black) and well-segmented masks (red). f: Scatter plot of gastruloid volume versus total cell count obtained by two independent methods. Blue: chemical dissociation and 2D volume reconstruction (for gastruloids dissociated after Chi-pulse only). Green: 3D segmentation for volume and cell count measurement (well-segmented cells only, see e). Slope of blue and green lines correspond to the mean \({V}_{c}\) for chemically dissociated and 3D segmented gastruloids, respectively. Upper left inset shows a close-up for small \(V\) and \(N\). Lower right inset shows correlation of variability for \(V\) and \(N\) for both methods. Note that the main error attached to the 3D segmentation volume is due to the estimation of the shrinkage factor of the mounting medium used in the phalloidin staining protocol (Extended Data Fig. 6C). 2D volume reconstruction from dissociated gastruloids is applied to images of live gastruloids (that is, they are not shrunken). g: Distribution of the logarithm of single cell volumes \({V}_{c}\) obtained by 3D segmentation after filtering and reconstruction for 96 h (\(n=28\)) and 120 h (\(n=20\)) old gastruloids with \({\overline{N}}_{0}=100\). Inset shows dispersion self-similarity \({{\rm{\delta }}}_{S}\), defined as \(\left\langle {\sigma }_{\log \left({V}_{C}\right)}/\overline{\log \left({V}_{C}\right)}\right\rangle\) for each set of distributions. It demonstrates the reproducibility of the dispersion in cell size in individual gastruloids and a further reduction in gastruloid cell size during the elongation process. The low variability indicates that the dispersion is highly conserved across gastruloids. h: Distribution of the effective cell diameter per gastruloid, obtained by chemical dissociation (only data from gastruloids dissociated after Chi-pulse) and 3D segmentation, overlaid by a Gaussian fit for each distribution. Vertical dotted lines correspond to the mean of each distribution. With the dissociation protocol, \({d}_{c}=13.9\pm 0.5\) μm (3.8%, \(n=286\)). With the 3D segmentation method, \({d}_{c}=13.1\pm 0.5\) μm (4.0%, \(n=108\)). Taking into account the different sources of error and our two independent methods of determination of the effective cell diameter, the relevant linear size of the system at 120 h is \({d}_{c}=13.5\pm 0.8\) μm. All error bars are standard deviations.