Extended Data Fig. 8: Characterization of E8.75 gut tube anterior–posterior pseudo-space.

a, Force-directed layout as in Fig. 5. Top, plots show the probabilities of anterior–posterior positioning for the AFP–GFP-positive and AFP–GFP-negative cells inferred using the manifold classifier trained on anterior–posterior cells. Bottom, plots show the probabilities of GFP-positive and GFP-negative status for the cells from the anterior–posterior compartment, inferred using the manifold classifier trained on GFP-positive and GFP-negative cells. b, Top, anterior and posterior cells labelled by measured data (leftmost two columns). Anterior and posterior positions of AFP–GFP-positive and AFP–GFP-negative cells inferred (rightmost two columns) using probabilities in a (top panels). Bottom, GFP-positive and GFP-negative cells labelled by measured data (leftmost two panels). GFP-positive and GFP-negative status of the anterior–posterior compartment cells inferred (rightmost two panels) using probabilities in a (bottom panels). c, Top left, plot showing the first diffusion component of the E8.75 cells. Top middle, top right, plots showing the expression of anterior marker Nkx2.1 and posterior marker Hoxb9 in E8.75 cells. Bottom, bulk RNA-seq expression of Nkx2-1 and Hoxb9 in quadrants of the gut tube along the anterior–posterior axis compares with anterior–posterior single-cell expression patterns. d, Plot showing the proportion of anterior and posterior cells in bins along the anterior–posterior pseudo-space. e, Heat map showing Pearson’s correlations between anterior–posterior pseudo-space orderings, determined using a varying number of diffusion components and highlighting the robustness of the ordering. f, Plots comparing the anterior–posterior pseudo-space ordering of GFP-positive and GFP-negative cells (replicate 2, 13,335 cells) generated de novo using only the replicate 2 cells (x axis, left) with the projected ordering from replicate 1 (8,143 cells) (y axis). Right, similar comparison with the pseudo-space ordering determined using cells of both the replicates on the x axis. g, Same as f, for replicates of anterior–posterior cells (replicate 1, 1,821 cells; replicate 2, 1,691 cells). Plots show the Pearson’s correlation. h, ROC for classification of E7.5 visceral and definitive endoderm cells (4,378 cells). i, Plots showing the expression patterns of genes that are best predictive of the definitive endoderm class in the visceral and definitive endoderm classifier (top, definitive endoderm; bottom, visceral endoderm). j, Plots showing the expression patterns of genes in the definitive endoderm that are best predictive of visceral endoderm class in the visceral and definitive endoderm classifier. k, Force-directed layouts following Harmony of E7.5 and E8.75 visceral and definitive endoderm cells, with E7.5 cells coloured in red (definitive endoderm) and blue (visceral endoderm) (left). E7.5 visceral and definitive endoderm cells coloured by the branch probability of anterior localization (middle) and posterior localization (right). Black arrowheads indicate early emergence of anterior–posterior spatial patterning at E7.5, with E7.5 definitive endoderm cells predominantly destined towards the anterior, and visceral endoderm cells predominantly destined towards the posterior. l, Three-dimensional renderings of gut tube, depicting all endoderm cells along the anterior–posterior axis. Nuclei of visceral and definitive endoderm cells are labelled in green and grey, respectively. m, Plots comparing the ranks of proportion of GFP-positive cells along anterior–posterior positioning in the AFP–GFP-embryo-derived Neurolucida reconstructed gut tube replicates (x axis), and the ranks of visceral endoderm cell proportions in bins along the anterior–posterior pseudo-space axis (y axis); the anterior–posterior axis was partitioned into 20 bins; each dot represents the fraction of visceral endoderm cells in that bin.