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Fig 2. Unsupervised analysis of variance between samples using principal component analysis (PCA) retrieves biologically meaningful components, matching embryo polarity axes, neural border (NB) formation, and developmental stage. (A, B) At Nieuwkoop and Faber stage (St.) 12.5 (A, circles) and St. 14 (B, triangles), the first 2 PCA components segregated ectodermal samples according to their position along the dorsal-ventral (D-V) and anterior-posterior (A-P) embryonic axes, respectively. Percent of total gene expression variance captured by each component is indicated between parentheses. Sample color code is identical to Fig 1A. Lighter background colors are Voronoi diagram areas. These areas are drawn using the barycenter of samples belonging to the same dissected region. Each area indicates the regions of the PCA plane, where all points are closer to the barycenter of this dissected region than to any other barycenter. Arrows indicate anterior neural border samples falling slightly outside their intended dissected region (see text for details). (C, D) At St. 12.5 (C) and St. 14 (D), t-distributed stochastic neighbor embedding (tSNE) groups samples according to dissected regions. (E, F) At St. 12.5 (E) and St. 14 (F), neural border samples are located higher than most others along the third PCA component. This indicates that component 3 correlates with the acquisition of the NB characteristics during neurulation. (G, H) When combining St. 12.5 (circles) and St. 14 (triangles) samples together, the first component remains correlated to the D-V axis. The second component sorts out most samples according to their stage, irrespective of their position along the D-V axis (triangles separated from circles, (G). The first and third PCA components segregate most samples according to their position along the D-V and A-P axes, irrespective of their stage (circles and triangles of same color together, [H], Voronoi diagram). The posterior neural border (NBp), which is known to resemble the posterior neural plate (NPp) at St. 12.5, grouped with the NPp, while the anterior neural border (NBa)–St. 12.5, NBa–St. 14, and the lateral neural border (NBl)–St. 12.5 belong to the same Voronoi cell. As noticed above, NBa–St. 14 is more heterogeneous (red triangles). Image published in: Plouhinec JL et al. (2017) © 2017 Plouhinec et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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