Fig. 4

Single-cell RNA-seq identifies two molecularly distinct populations of PSNs prior to cell death period. a Workflow for single-cell RNA-seq (scRNA-seq) processing of DRG neurons. b Heat map showing single-cell expression of the top 20 differentially expressed genes in the different clusters of DRG neurons, identifying Clusters 1 and 2 as PSNs. c t-SNE plot showing two distinct populations of Ntrk3⁺/Runx3⁺ PSNs at E11.5, the Clusters 1 and 2 in b. eTRKA: early TRKA neurons; MR: mechanoreceptive neurons¹⁸; both populations transiently express TRKC at an earlier time point³⁸. d List of Cluster 1- (top panel) and Cluster 2-enriched (bottom) genes. e The two clusters express similar levels of classic neuronal markers but differ in their level of Runx3 and Ntrk3 transcripts and are named Runx3^High and Runx3^Low. Box plots visualize the summary of the dataset (minimum, lower quartile, median, upper quartile and maximum). Wilcoxon rank sum test (# indicates significant difference, see Methods section for details). f, g In vivo validation of the correlation between TRKC protein and Runx3 mRNA (f) and between TRKC protein and Ntrk3 mRNA (g). ^**P < 0.01, unpaired Student’s t-test (n = 3). h–j Marker genes of the Runx3^Low and Runx3^High subgroups of PSNs; t-SNE plots (h) and in vivo validation by immunostaining or in situ hybridization (i). Graphs in i show quantification of the expression of marker genes for Runx3^High (High) and Runx3^Low (Low) PSNs (n = 3 animals for each experiment), expressed in average number of dots for transcripts and average immunostaining intensity for proteins (and normalized to the maximum average value). ^***P < 0.001, unpaired Student’s t-test. Scale bar: 5 μm. j In situ hybridization on E11.5 DRG sections showing the (almost) mutually exclusive expression of Fxyd7 and Pou2f2. Scale bar: 15 μm. k Quantitative illustration of j, where for each TrkC⁺ neurons, their level of Fxyd7 and Pou2f2 is represented. l Schematic representation of neuronal types with their key markers and their average soma size (in square micrometers). Source data are available as a Source Data file