Fig. 2. Size-dependent emergence and resolution of multipolarity.

(A) Representative images of Mesp2-mCherry expressing gastruloids at 72, 96, 120, and 144 hours across varying N₀. Scale bars, 200 μm. (B) Heatmap of Mesp2-positive pole counts (0 through 4, top legend) over time for gastruloids with N₀ = 150 (n = 41), 300 (n = 49), 600 (n = 48), and 1200 (n = 51). Each vertical line represents a gastruloid (see fig. S2D). (C) Cartoon showing unpolarized, polarized, and uniaxial gastruloids; arrows indicate flow of time. Left: Maximum Mesp2 poles per gastruloid. Right: Fraction of gastruloids that were multipolar at least once and uniaxial at the end (>98%). (D) Temporal dynamics of Mesp2 poles (means ± SEM) for different N₀. (E) Top: Histograms of polarization time points (transition from 0 to ≥1 Mesp2 poles). Bottom: Histograms of uniaxial gastruloid formation (transition to one stable Mesp2 pole). Solid lines indicate Gaussian fits (see supplementary tables for statistics). Scatterplot (right) shows means ± SEM of these time points versus gastruloid volume. Gray dotted lines show linear fits of the means to guide the eye. (F) Maximum (left) and minimum (right) distances between peak local maxima of the Mesp2 poles as functions of multipolarity resolution time (measured as the transition time point from >1 Mesp2 pole to a single stable pole). Gray solid line indicates the average minimum distance across all conditions (min = 253 ± 77 μm).