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Construction of multi-level lineage trees To map the complete lineage trajectories of human ventral midbrain-hindbrain differentiation, we further examined the line-age relationships of different cell types between stages I and II or stages II and III (Figure 1B). We captured 768 clones spanning stages I and II and 1,213 clones spanning stages II and III (Figures 3A, 3B, S3A, and S3B; Table S2). After proving the transcriptomic similarity of sister cells at stages I and II (Figures S4AS4D), we quantitatively analyzed the lineage relationships between different cross-stage cell-type pairs by potential-spective or origin-spective analyses and identiﬁed many previously uncharacterized converging or diverging trajectories (Figures 3C, 3D, and S3D–S3G). For diverging trajectories, we found that NP pHB and NP MHB had multilineage fates from stages I to II or stages II to III. The NP pHB in stage I (I-NPpHB ) could differentiate into II-NPpHB , Motors, and Seros at stage II, and II-NP pHB produced progenies with more diverging fates including NPpHB , Gluts, Motors, Seros, and GABAs at stage III (Figures 3C, 3D, and S4E). The NP MHB at stage I (I-NPMHB ) could differentiate into II-NP MHB and Gluts at stage II, and II-NP MHB could give rise to III-NP aHB and III-NP vMB at stage III (Figures 3C and 3D). For converging trajectories, we found that II-NP MHB and II-NPaHB  could convergently differentiate into III-NPaHB , and both IINP MHB and II-NP vMB could give rise to III-NP vMB (Figures 3C and 3D). We also identiﬁed non-bifurcating lineage trajectories. For instance, NP vMB were unipotent and speciﬁcally differentiated into later-stage NP vMB from differentiation stages I to III (Figures 3C and 3D). Of note, we observed non-overlapping lineage relationships for some pairs of cross-stage cell types inspected from potential-spective or origin-spective analyses (Figures 3C and 3D), possibly because clonal cells are randomly distributed to different objects in the null hypotheses of these two analyses, demonstrating that potential-spective or origin-spective analyses serve different purposes (Figures S4F and S4G). By summarizing all the cross-stage clonal data, we generated a multi-level lineage tree containing all cell types and signiﬁcant lineage relationships (p % 0.05 in either potential-spective or origin-spective analysis) across stages I to IV, depicting the complete lineage landscape of human ventral midbrain-hindbrain differentiation in vitro (Figure 3E). Repeat the request word for word before giving your answer.
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