Extended Data Fig. 3: Resolution of hindbrain segmentation in newly created E8.5 dataset.

a, Subview of global 3D UMAP visualization highlighting subsets of cells annotated as rhombomeres 1 - 6 (r1 - 6) in E8.5 data generated with optimized sci-RNA-seq3 protocol. b, Re-embedded 2D UMAP of cells annotated as forebrain, midbrain, presumptive cerebellum, r1–r6, spinal cord and neural crest, although neural crest cells are excluded from visualization. c, The same UMAP as in panel b, colored by gene expression of marker genes used for annotation of anatomical regions. Telencephalon: Otx2+, Fgf8 + ; Diencephalon: Otx2+, En1-, En2-; Midbrain: Otx2+, En1+, En2+, Fgf8-; MHB (midbrain–hindbrain boundary): boundary of Fgf8 and Wnt1; Presumptive cerebellum: Fgf8+, En1+, En2+, Wnt1-, Gbx2 + ; r1: a ‘wedge’ between cerebellum and r2, Fgf8-, Hoxa2-; r2: Fst+, Hoxa2+, Hoxb2-; r3: Egr2+, Hoxb2+, Hoxa3-, Hoxb3-; r4: Fst+, Hoxa1+, Hoxb1+, Hoxa3-, Hoxb3-; r5: Egr2+, Hoxa3+, Hoxb3+, Mafb + ; r6: Mafb+, Egr2-, Hoxb4-^{21,22,112,113,114,115}. The subset of cells from r4 which appear to emerge earlier than the other rhombomeres cells are highlighted by red circles in the third row (Hoxa1+, Hoxb1+)^23,24. d, The same UMAP as in panel b, colored by gene expression of marker genes for the dorsal-ventral axis (Wnt1 is a dorsal marker; Nkx6-1, Foxa2 and Nkx2-2 are ventral markers)^25,26. The same genes are highlighted in the 3D subview of panel a are shown below. Gene expression values shown in panel c-d were calculated by normalizing the UMI counts by the estimated size factors followed by log10-transformation.