Extended Data Fig. 2: Antero-Posterior patterning in Somitoids.
From: Reconstruction and deconstruction of human somitogenesis in vitro
a, Illustration of the design of the HES7/MESP2/UNCX triple-reporter cell line. b, Ratio of mean mCherry or YFP intensities in the centre circle vs in the big circle (n = 8 Somitoids and the bars indicate median). c, Normalized RNA counts of selected polarity genes in cell fractions separated by flow cytometry, as measured by RNA sequencing (n = 3 independent experiments, 96 Somitoids in each n). Cells with top 10% mCherry fluorescence are shown on the left (magenta) and top 10% YFP fluorescence on the right (yellow). All four genes were identified as differentially expressed genes by DESeq2 using the Wald test (two-sided). d, Temporal plot of HES7 reporter (mean±s.d., n = 3 Somitoids) and images of an UNCX and MESP2 reporting Somitoid treated with 50 µM DAPT added at 48 h. e, Wide-field images of PAX3-reporting Somitoids treated with 50 µM DAPT (left) since 48 h and control (right). f, Maximum-z-projection confocal images of UNCX and MESP2 reporting Somitoids treated with 10 µM ROCKi (left) or 5 µM Blebbistatin (right) since 48 h. g, Left, percentage of UNCX-positive cells characterized by flow cytometry in 120 h WT (n = 6 experiments), HES7-null (n = 6 experiments), and MESP2-null (n = 5 experiments) Somitoids; Data are represented as mean±s.d., one-way ANOVA, compared with WT, P = 0.89 (HES7-null); 2.49e-10 (MESP2-null). Right, images of MESP2 and UNCX reporters in HES7-null Somitoids, and UNCX reporter in MESP2-null Somitoids. h, Histograms of flow cytometry analysis of UNCX-YFP in 120 h Somitoids (control, WT, HES7-null, and MESP2-null cell lines) with debris and doublets removed. Control is the parental NCRM1 cell line. Fractions on the right side of the red dotted line in the histograms are defined as YFP-positive. i, Scattered plot (top) and histogram (bottom) of flow cytometry analysis on MESP2-mCherry Somitoids at 72 h with debris and doublets removed. j, Time-lapse images of MESP2 reporter in a Somitoid. k, Time-lapse maximum-z-projection confocal images of H2B-GFP in the same region of a Somitoid as in Fig. 2d. l, Cell tracks of MESP2-high cells overlayed on images of MESP2 reporter. The orange outlines represent the forming MESP2-low regions. m, Spatial auto-correlation (sole MESP2 signal, sole UNCX signal or them combined together) once rosettes are formed (representative example from n = 3 Somitoids). n, Additional example of spatial auto-correlation analysis and abscissa-position of the auto-correlation trough (inset) of MESP2/UNCX double reporting Somitoid over time. o, Temporal plot (mean±95%CI) of mean squared displacement (n = 3,422 tracks from 2 Somitoids). p, Additional example of normalized temporal profiles of MESP2 reporter in individual cells (top; n = 52 cells from one Somitoid), and correlation analysis of MESP2 intensities at 72 h and 84 h (bottom; F-test, one-sided, P = 4.67e-14 after removing 3 outliers identified by calculating Mahalanobis distance, as explained in Methods, marked by magenta cross). Temporal profiles are coloured based on relative MESP2 intensity among tracked cells at 72 h, with higher 50% in magenta and lower 50% in cyan throughout the time window. q, Surrounding MESP2 intensity (Methods) of tracked cells at 72 h and 84 h (n = 98 cells from two Somitoids; unpaired two-tailed t-test). Cells at both time points are grouped based on relative MESP2 intensity at 72 h, with lower 50% on the left (cyan) and higher 50% on the right (magenta). r, Left, temporal profile of MESP2 intensity in cells starting in a correct (orange) or wrong (green) region (Method). Right, end-time-point MESP2 intensity of cells with correct or wrong start. n = 98 cells from two Somitoids; unpaired two-tailed t-test. s, Left, temporal profile of displacement in cells starting in a correct (orange) or wrong (green) region (Method). Right, end-time-point displacement of cells with correct or wrong start. n = 98 cells from two Somitoids; unpaired two-tailed t-test. t, Velocity field (arrows) and the corresponding divergence (heatmap) of Particle Image Velocimetry analysis (left) on an additional Somitoid and regions of positive divergence overlayed on the MESP2 reporter image (right; yellow outlines). u, Summary of MESP2 expression and pattern formation processes in the timeline of the Somitoid differentiation. v, Quantification of UNCX reporter in MESP2-high (n = 8 re-aggregates from 3 experiments) and MESP2-low (n = 6 re-aggregates from 3 experiments) re-aggregates in Fig. 2l-n, paired two-sided t-test. In all box and whisker plots, the centre indicates the median, the upper bound indicates 75th percentile, and the lower bound indicates 25th percentile. The maxima and minima of the whiskers represent the most extreme non-outlier data points. The outliers are defined as data points greater than the upper bound or smaller than the lower bound by more than 1.5 times the interquartile range. Scale bars represent 500 µm (b, d, f, g, j), 200 µm (e, t), and 100 µm (k, l).
