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. 2023 Jun 1;30(6):867–884.e11. doi: 10.1016/j.stem.2023.04.018

Figure 3.

Figure 3

Mapping spatial cell-type emergence with image-based trajectory

(A) Scheme illustrating automatized handling workflow. Right: representative image of gastruloids fixed at 120 h. Maximum intensity projection (MIP) of z stack: DAPI and antibody stainings for Sox2 and Bra. Scale bars, 150 μm.

(B) Scheme illustrating extracted features and super pixel analysis.

(C) Representative images at indicated time points (middle z plane of a z stack showing DAPI, Bra, and Sox2. Scale bars, 100 μm. Hexbin plots: mean normalized superpixel intensities of Sox2. Kernel density plots: distribution of intensities along the x and y axes; dist. to center, normalized distance of superpixel to object center (bottom). Co-expression hexbin plots: expression of Sox2 and Bra. Kernel density plots: distribution of intensities along x and y axes (right). Sample numbers (n) are indicated.

(D) UMAP plots of n = 2,862 gastruloids color-coded by time points. Bottom left: scheme illustrates increasing heterogeneity in later time points.

(E) Inferred pseudotime (top left) and scheme of pseudotime ordering, trajectory inference, and molecular progression.

(F) Heatmaps depicting distribution of stainings from the anterior (left) to the posterior (right) pole along pseudotime (progressing from bottom to top). n, number of gastruloids. Anterior bias (light green), posterior bias (light red), and unbiased markers (light yellow).

(G) Representative images at 96 h. (MIPs of z stack showing DAPI and antibody stainings for Oct4, Cdx2, Eomes, Hes1, Foxa2, Sox1, E-Cad and N-Cad, and Sox2). Scale bars, 100 μm.

(H) Top: representative images at indicated pseudotime and sampling time points. Middle z plane: DAPI and antibody stainings for Fn1 and Sox2. Scale bars, 100 μm. Bottom: heatmaps depicting distribution of Fn1 and Sox2 staining from anterior (left) to posterior (right).

(I) Top: line plots of mean staining intensity for Sox2 and Fn1 (n = 82). Blue bars: individual gastruloids shown in (H). Middle: heatmaps depicting Fn1 distribution from anterior (top) to posterior (bottom) along pseudotime. Bottom: heatmap depicting Sox2 and Fn1 in/out ratio measured on the middle z plane.