Extended Data Fig. 5: scRNA-seq tissue assignment and wild-type comparison.

a, Box plots representing tissue proportions from E8.0 (top) and E8.5 (bottom) wild-type embryos (n = 10 each), with lineage-traced embryos mapping to each state overlaid as dots. Wild-type embryos display a large variance in the proportions of particular tissues, and the proportions of our lineage-traced embryos generally fall within the range of those recovered from wild type. Large circles indicate embryos that were scored as either E8.0 or E8.5, and the bold red overlay highlights embryo 2, which is used throughout the text. Note that many processes—such as somitogenesis and neural development—are continuous or ongoing between E8.0 to E8.5. For example, from E8.0 to E8.5, the embryonic proportions of anterior neural ectoderm and fore- and midbrain are inversely correlated, as one cell type presumably matures into the other. Many of our embryos scored as E8.0 exhibit intermediate proportions for both tissue types, which supports the possibility that these embryos are slightly less developed than E8.5 but more developed than E8.0. For box plots, the centre line indicates the median, edges indicate the interquartile range, whiskers indicate the Tukey fences, and crosses denote outliers. b, t-SNE plots of scRNA-seq data with corresponding tissue annotations for the six lineage-traced embryos used in this study. Insets, pie charts of the relative proportions for different germ layers. Mesoderm is further separated to include blood (red). Although 36 different states are observed during this developmental interval, only broad classifications of particular groups (for example, neural ectoderm or lateral plate mesoderm) are overlaid to provide a frame of reference. In general, the relative spacing and coherence of different cell states are consistent across different embryos. c, Box plots of the Euclidean distance between single-cell transcriptomes and the average transcriptional profile of their assigned cluster (cluster centre) in comparison to their distance from the average of the next-closest possible assignment. Comparison is to the same 712 informative marker genes that were used to assign cells to states, and includes all cells used in this study (Supplementary Methods). Middle bar highlights the median, edges indicate the interquartile range, whiskers indicate the Tukey fences, and grey dots denote outliers. n values refer to the cumulative number of cells assigned to each state across all seven embryos for which single-cell data were collected, including for embryo 4, which was ultimately withheld from further analysis owing to the lack of primitive heart tube development.