Differentiation trajectories of FRCs and VSMCs in murine lymph nodes. (A) Schematic representation of the workflow for transcriptome analysis of FRCs from mesenteric lymph nodes of Ccl19-iEYFP mice using droplet-based scRNA-seq. (B and C) UMAP visualizing Ccl19-iEYFP⁺ cells from mesenteric lymph nodes colored by (B) age group and (C) FRC subset identity derived from the collective analysis of all adult FRCs. (D and E) UMAP visualizing Ccl19-iEYFP⁺ cells from mesenteric lymph nodes with the inferred differentiation lineages from slingshot analysis (D) and cells colored by the inferred slingshot pseudotime (E). (F) Expression fits of the assigned genes along the pseudotime for each of the inferred slingshot lineages. Genes that have similar expression patterns along all lineages were clustered, and clusters with >7 genes are shown. (G) Selected differentially expressed genes in the slingshot TRC/BRC/MedRC lineages along the pseudotime. (H) Selected differentially expressed genes in the slingshot PRC/VSMC lineages along the pseudotime. (I) UMAP visualizing Ccl19-iEYFP⁺ cells from mesenteric lymph nodes colored by cluster identity inferred from unbiased clustering. (J) Heatmap showing the scaled average activity of transcription factors (TFs) across clusters of Ccl19-iEYFP⁺ cells from mesenteric lymph nodes. For each adult FRC/VSMC cluster the top five transcription factors with the highest averages activity are shown. (K) Scaled activity in each cell of the most active transcription factors for adult FRC/VSMC clusters projected on the UMAP of Ccl19-iEYFP⁺ cells from mesenteric lymph nodes. Mesenteric lymph node scRNA-seq data are representative of n = 10–15 mice per time point; 33,903 cells from E18; 10,005 cells from P7; 12,291 cells from 3-wk-old mice; 52,188 cells from 8-wk-old mice.