Extended Data Fig. 3: Immunofluorescence image analysis.

a: Fixed gastruloids are imaged by confocal microscopy in z-stacks of 150 μm (30 slices, dz = 5 μm). b: Analysis pipeline of Extended Data Fig. 1c is applied to the DAPI channel for each gastruloid to extract midline, contour, and equidistant slices. Fluorescence intensities of the other channels are max projected (here illustrated with SOX2 (green) and CDX2 (red)) and intensities of individual slices are integrated to obtain a single value per slice and to construct one-dimensional expression profiles as a function of slice position along the midline. Scalebar is 100 μm. c: One-dimensional profiles of SOX2 (green) and CDX2 (red) along the midline obtained for the gastruloid in b. d: Visual comparison of mean (left) versus maximum (right) projection of a gastruloid stained for SOX2 (green) and CDX2 (red). Scalebar is 100 μm. e: Quantitative comparison of maximum (x-axis) versus mean (y-axis) projection of intensities for the four examined genes in individual gastruloids from Fig. 2 (\(n=\){44, 44, 48, 46} respectively for SOX2, CDX2, BRA and FOXC1). Color code corresponds to the position of each slice along the midline (yellow towards the anterior pole, gray-blue towards the posterior pole). f: Mean profiles of expression of the four genes as a function of relative position \(x/L\) using either maximum (black) or mean (gray) projection. g: Variability as a function of the relative position \(x/L\) along the midline of each set of gastruloids for the four genes. Gray and black lines correspond to the variability computed respectively from either mean or maximum projections. Measured variability is lower when using maximum projection. h: Visual comparison of gastruloid slicing methods, straight lines (left, yellow) versus curved lines (right, pink); immunostained gastruloid stained for SOX2 (green) and CDX2 (red). Straight lines are line segments calculated between the equidistant points along both sides of the contour as in Extended Data Fig. 1c. Curved lines are obtained using both equidistant points along the contour and along the midline. From this combination of points, a parabolic equation is calculated using a second-order polynomial fit. This procedure is meant to recapitulate the overall curvature of the gastruloid. i: Quantitative comparison of intensities using straight (x-axis) versus curved (y-axis) line slicing for the four examined genes in individual gastruloids from Fig. 2 (\(n=\) {44, 44, 48, 46} for SOX2, CDX2, BRA and FOXC1, respectively). Color code corresponds to the position of each slice along the midline (yellow towards the anterior pole, gray-blue towards the posterior pole). j: Mean profiles of the four stained sets of gastruloids from Fig. 2 as a function of relative position \(x/L\) using either straight (yellow) or curved (pink) line slicing. k: Variability as a function of the relative position \(x/L\) along the midline of each set of gastruloids for the four genes. Yellow and purple lines correspond to straight and curved line slicing, respectively. Using the curved lines method diminishes border effects on profiles of the four genes (mean and variability). No significant change is observed for the most part of the gastruloid midline, making both methods essentially equivalent. For computational simplicity, we employ the straight lines method. All profiles are represented for \(0.1\le x/L\le 0.9\) in the rest of the paper.