Extended Data Fig. 1: Single cell analysis of ApjCreER lineage labelled cells.

a, Comparison of rPCA and classical PCA at separation of subpopulations. PC scores were selected to best separate the Enpp⁺ Esam⁻ population. Cells are coloured by expression (log₁₀ CPM, scaled to maximum per gene). n = 352 cells. b, Comparison of default and sum-of-60 modified PC scores. PC2 is the default PC score from rPCA; PC2.score is the modified sum-of-top-60 scores (expression is log₁₀ CPM, scaled to maximum). Y-axis is the number of genes detected per cell (>1 count). n = 426 cells. c, Comparison of default and sum-of-top-60 scores. Scores were chosen that best separated the Vwf⁺ and Cxcr4 populations^. n = 426 cells. d, Unique cell cycle signature on PC.pos/PC.neg biplots. PC1.pos (PC1.neg) is the sum of the top 30 genes by positive (negative) loading to PC1. Cells are coloured by expression. Lower panel is the same rPCA after removing the list of 202 cell cycle genes. Numbers in bold are the correlations between PC1.pos and PC1.neg. n = 674 cells. e, PC.pos/PC.neg biplot showing theoretical location of doublets expressing high levels of both gene sets. f, Schematic of the pairwise discreteness test on a discrete (left) and continuous (right) pair of subpopulations. g, FACS plots used to isolate GFP-positive cells (red box) from ApjCreER Rosa^mTmG hearts at E12.5. h, Top, discreteness statistic generated by pairwise discreteness test as a function of number of intermediate cells (n_int) for simulated distributions. Bottom, pairwise distributions of cell clusters in the data set and the fraction of intermediate cells estimated by pairwise discreteness analysis. i, rPCA plots and their accompanying gene expression patterns in the embryonic heart as reported by Euroexpress. In situ hybridization images show whole hearts (top); insets of specific areas are in lower panels with relative expression levels indicated. Expression levels in rPCA plots range from 0 (yellow) to 4 (brown) in log₁₀CPM. Top, n = 843 cells. j, Summary of broadly defined cell populations as indicated by gene expression patterns. n = 843 cells. k, Example of manual clustering process. For i, n = 732 cells; ii, n = 531 cells; iii, n = 415 cells; iv, n = 284 cells; v, n = 261 cells. l, Comparison of pairwise discreteness test results for different numbers of genes per cell type signature (n).