Fig. 4: iTracer temporal lineage recording illuminates a window of fate restriction in cerebral organoids associated with brain patterning.

a, Lineage plot shows full lineage reconstructions from a single organoid scarred at day 15 and sequenced at day 63 (left) as well as the subset of cells in which scars were detected (right). First- and second-order deviation nodes represent barcode and scar families, respectively, with terminal branches indicating individual cells. Each cell is colored based on the cell type designation. b, t-distributed stochastic neighbor embeddings (t-SNE) show cells for this organoid colored by both cell type annotation and scar families (≥5 members). c, Force-directed graph embedding of cells from a single barcode family within organoid 1 (Org1), with cells colored by scar family or cell type (inset). Scar families in orange show enrichment in cortical brain regions. d, Frequency distribution of z scores of scarring pattern distances between cell clusters after subtraction of background distribution estimated by random sampling of scars. Different scarring times are shown separately. Dashed shadow backgrounds show 90% confidence intervals, accordingly colored by scarring time. This plot highlights that scarring at later time points separates lineages with different cell fates. e, UMAP embedding of developing cerebral organoids scarred at day 7 and sequenced at day 15, with cells colored by cell types and estimated brain regional identity or samples (inset). RG, radial glia; NE, neuroepithelial. f, Expression of regional marker genes and selected morphogen genes. g, Heatmap of relative similarity of organoid clusters to organizing centers identified in developing mouse brains. T, telencephalon; D, diencephalon; M, mesencephalon; R, rhombencephalon; Hb., hindbrain; Mb., midbrain; Fb., forebrain; PCC, Pearson’s correlation coefficient. h, Lineage plot shows full lineage reconstructions from one day 15 organoid (sample 1). The zoomed-in view highlights a barcode family with diverse patterning states. i, Bar plot showing region composition of scar families, revealing that cells are not yet restricted at the time point of scarring at day 7. P values show results of two-sided Fisher’s exact tests to compare regional proportions in different scar families, with or without consideration of unscarred cells.