Imaging and quantification of embryo development.

(a) MIP image of an embryo at 50% epiboly; a sequence of four time-lapse images from a selected region (square) is shown on the right. The arrows point to a dividing cell. (b) Typical maximum intensity projection images of a zebrafish embryo at 2.5, 5, 8, 11, 14, and 17hpf. Scale bar is 100µm. (c) Time dependence of the average cell number and yolk surface coverage during embryo development, computed from an ensemble of five embryos.

Alignment of cell coordinates according to the standard embryo position.

(a) Standard embryo representation on the yolk sphere at later developmental time points (16hpf); the color code denotes the distance from the origin. (b) Transformation from Cartesian to spherical coordinates. For the cylindrical projection, the anterior-posterior embryo axis was placed at zero elevation (equator); by setting the dorsal embryo side at zero azimuths, the animal (AP) and vegetal (VP) poles appeared at the π/2 and π/2 azimuthal angles, respectively. (c) Embryo alignment at the early gastrula stage. Top and side views show the symmetrically distribution of cells around the AP. The color code denotes the distance from the origin as in (a). The radius of the embryo sphere R₀ is determined according to the border between hypoblast and epiblast cell layers (lower panel of the side view; the color code denotes the movement direction) which display opposite movement at 7hpf towards the AP (red) and VP (blue), respectively. (d) Spherical projection of cell nuclei coordinates from a single embryo on a 2D map using azimuthal and elevation angles. Typical 2D maps of cell nuclei coordinates are plotted at 6, 8.5, 10.5 and 16hpf using the equidistant cylindrical projection. The radial coordinate is color-coded in the range between 0.8 and 1.4. (e) 2D maps of integrated cell nuclei density from a single sample using the area-preserving Gall-Peters (2 sinθ versus Φ) and R versus Φ projection of the same datasets as in (d). The blue-to-red color code denotes the increase of the normalized cell density in arbitrary units.

Ensemble-averaged digital model.

(a) Averaged cell density cylindrical projections in spherical coordinates (azimuth vs. elevation). The vector fields represent 2h collective cell migration patterns, computed by locally averaging the displacement of cells with R < 1 (red arrows) and R > 1 (dark cyan). For symmetry reasons, only half of each vector field is shown. (b) Planar projections in Cartesian coordinates at selected times; the dashed white line indicates the embryo sphere, and the blue-to-red color code represents the normalized cell density (in arbitrary units). (c) Projection of cell density movement at the embryo dorsal side from 6 to 8.5hpf; the direction is encoded by color (toward the AP: red, toward the VP: blue). (d) Cell density projection at 16hpf at a cross-section through the head region (d–d2) depicted in panel (b); the dashed black lines mark the eye positions. The color code is the same as in (a) and (b). (e) Kymographs of azimuthal (left panel) and elevation (right panel) cell density projections; the color code denotes the number of cells per azimuthal or elevation segment, respectively. (f) Quantitative analysis of cell density (upper panel) and movement (lower panel) variations with respect to the midline mirror (left-right) symmetry (within each sample) and to the model. The solid lines represent averages over the ensemble; the shaded regions around the lines depict the ±σ values of statistical significance that include sample heterogeneity as well as statistical errors due to cell number variations within voxels (σ² = (σ_samples)² + (σ_ΔN )²).

Visualization of blastoderm margin development.

(a) Lateral view of a zebrafish embryo at 6hpf; cells are shown as small gray dots. The colored spheres denote examples of hypoblast (red) and epiblast (blue) cells; also shown are their trajectories from 6–10hpf. (b) Typical trajectories of selected hypoblast (left) and epiblast (right) cells during gastrulation; represented by their positions projected onto the AP-VP axis (top) and their radial positions (bottom). Trajectories are color-coded according to dorsal, lateral or ventral position; the dashed line (upper graphs) represents the position of the blastoderm margin. (c) Gall-Peters projections and radial plots of cell positions, showing an overlay of hypoblast (left) and epiblast (right) cells from different samples (shown in different colors) on top of the averaged model cell density (gray) at 9hpf. (d) Hypoblast (left) and epiblast (right) azimuthal cell density kymographs (yellow/red) overlayed onto the digital model kymograph from Fig. 3e (gray).

Morphological changes of an oep mutant.

(a) MIP image of an oep mutant embryo at 18hpf and cell density projection at 16hpf. Arrows indicate characteristic phenotypical oep features. (b) Quantitative comparison of wt and oep mutant cell number (top), epiboly (middle), and cell density variation during development (bottom). Data of the wt embryo (Fig. 3F and Supplementary Fig. 3) are included for comparison. (c) Left and middle panels: Cell density projections at 8hpf of the averaged model and the individual oep mutant, respectively, depicted in the same color code as in Fig. 3a. Right panel: Overlay of differences (negative: cyan, positive: red) in cell densities between the mutant and the wt model on top of the model data (gray). (d) Kymographs of azimuthal projections, panels arranged as in (c). Arrows point to pronounced developmental differences between the oep mutant and the wt embryo model.