Fig. 5: Characterisation of pathogenic enhancer variant activity at single-cell resolution.

a Single-cell transcriptomic profiling of E11.5 hindlimbs from transgenic mice in which hZRS^ref drives mCherry and hZRS^404G>A drives eGFP. A nested PCR strategy was used to amplify mCherry and eGFP reporter transcripts (Methods). b Integrated UMAP plot showing ~ 21,0000 cells clustered by cell type. Mesenchymal clusters are defined by the expression of spatially-defined genes. HSCs, hematopoietic stem cells. c Feature plots showing mCherry and eGFP expression with overlapping cells marked in yellow. Areas of ectopic eGFP expression are highlighted in magenta with an accompanying fluorescent image on the right. Data derived from one dual-enSERT-1 and one dual-enSERT-2 replicate. Scale bar, 500 μm. d Dot plot quantifying percentage and normalised expression of cells expressing eGFP, mCherry, and Shh within each cluster. Clusters with ectopic eGFP expression are highlighted. e Heatmap of differentially expressed genes between mCherry + (normal hZRS activity), mCherry-/eGFP + (ectopic hZRS activity), and mCherry-/eGFP- (inactive ZRS) cell subpopulations. Unsupervised hierarchical clustering of genes on the left; select marker genes on the right. f UMAP plots of reclustered mCherry + and eGFP + cells with accompanying feature plots depicting their expression. g Violin plots quantifying mCherry and eGFP expression across clusters. h Volcano plot depicts differential gene expression between Cluster 3 and the other clusters. Genes upregulated (Adjusted P-value < 0.05 and log₂FC > 2) in Cluster 3 are coloured in teal. Non-parametric Wilcoxon-rank sums test. The spatial distribution of representative marker genes in E11.5 limb buds is shown on the right. Images reproduced with permission from the Embrys database (http://embrys.jp).