Extended Data Fig. 7: Characterizing cellular trajectories during limb mesenchyme differentiation.

a, UMAP 3D visualization of limb mesenchymal cells coloured by development stage (cell number, n = 26,559; left and right represent views from two directions). b, Heat map showing top differentially expressed genes between different developmental stages for limb mesenchyme cells. c, Bar plot showing the −log10-transformed adjusted P value (one-sided Fisher exact test with multiple comparisons adjusted) of enriched transcription factors for significantly upregulated genes during limb mesenchyme development. d, t-SNE visualization of limb mesenchyme cells coloured by forelimb (Tbx5⁺; cell number, n = 2,085) and hindlimb (Pitx1⁺; cell number, n = 1,885). Cells with no expression or expression of both in Tbx5 and Pitx1 are not shown. e, h, i, k, Each panel illustrates a different marker gene. Colours indicate UMI counts that have been scaled for library size, log-transformed, and then mapped to Z-scores to enable comparison between genes. Cells with no expression of a given marker are excluded to prevent overplotting. e, Hindlimb marker Pitx1 and forelimb marker Tbx5. f, Scatter plot showing the normalized expression of Pitx1 and Tbx5 in limb mesenchyme cells. Only cells in which Pitx1 and/or Tbx5 were detected are shown. g, Volcano plot showing the differentially expressed genes (FDR of 5%, one-sided likelihood ratio test with multiple comparisons adjusted, coloured red) between forelimb (cell number, n = 2,085) and hindlimb (cell number, n = 1,885). Top differentially expressed genes are labelled. x axis, log₂-transformed fold change between forelimb and hindlimb for each gene; y axis, −log₁₀-transformed q value from differential gene expression test. h, Same visualization as e, coloured by normalized gene expression of proximal/chondrocyte (Sox6 and Sox9), distal (Hoxd13 and Tfap2b), anterior (Pax9 and Alx4) or posterior (Hand2 and Shh) markers. Only cells with the gene marker expressed are plotted. i, Same visualization as e. First row, proximal limb markers Sox6 (which also marks chondrocytes) and Sox9. Second row, distal limb markers Hoxd13 and Tfap2b. Third row, anterior limb markers⁶⁵ Pax9 and Alx4. Fourth row, posterior limb markers Shh and Hand2. j, In situ hybridization images of Hoxd13 in E9.5 to E13.5 embryos (n = 5). k, Same visualization as e, coloured by normalized gene expression of Cpa2. Only cells with positive UMI counts are shown. Values are log-transformed, standardized UMI counts. The expression pattern of Cpa2 within this trajectory led us to predict that it is a distal marker of the developing limb mesenchyme, similiar to Hoxd13. l, In situ hybridization images of Cpa2 in E10.5 and E11.5 embryos (n = 5. Arrow, site of gene expression. m, Modules of spatially restricted genes in the limbs. A total of 1,783 genes were clustered via hierarchical clustering. The dendrogram was cut into eight modules using the cutree function in R, and the aggregate expression of genes in each module was computed. Colours indicate aggregate UMI counts for each module that have been scaled for library size, log-transformed and then mapped to Z-scores to enable comparison between modules. Cells with no expression of a given module are excluded to prevent overplotting.