Differentiation trajectories of Ccl19-iEYFP ⁺ and Cxcl13-EYFP ⁺ FRCs and VSMCs in murine lymph nodes. (A) Sorting strategy of EYFP⁺ progenitors for the scRNA-seq analysis of lymph node anlagen from Ccl19-iEYFP⁺ and Cxcl13-EYFP⁺ embryos at E18. (B) Dot plot indicating the average expression of signature genes across embryonic Cxcl13-EYFP⁺ cell populations. (C–E) UMAP representation of Cxcl13-EYFP⁺ cell clusters (C) and projection of proliferation (D) and perivascular/mural (E) signatures consisting of the indicated genes on the scRNA-seq dataset. (F and G) Confocal microscopy images showing cross sections of inguinal (F) and mesenteric (G) lymph nodes isolated on postnatal day 7 from Ccl19-iEYFP⁺ pups. Microscopy images are representative for three inguinal and three mesenteric lymph nodes from three independent experiments. Scale bars: 20 µm (F) and 200 µm (G). (H–J) Confocal microscopy images showing cross sections of inguinal (H) and mesenteric (I) lymph nodes isolated on postnatal day 7 from Cxcl13-EYFP⁺ pups. Boxed areas indicate regions of higher magnification (J). Microscopy images are representative for three inguinal and three mesenteric lymph nodes from three independent experiments. Overviews in G and I are stitched images from tile scans. Scale bars: 50 µm (H), 200 µm (I), and 50 µm (J). (K) Schematic representation of the workflow for transcriptome analysis of EYFP⁺ cells from mesenteric lymph nodes of Cxcl13-EYFP mice using droplet-based scRNA-seq. (L and M) UMAP visualizing Cxcl13-Cre⁺ cells from mesenteric lymph nodes colored by (L) age group and (M) FRC subset identity derived from the collective analysis of all adult FRCs. (N and O) UMAP visualizing Cxcl13-Cre⁺ cells from mesenteric lymph nodes with the inferred differentiation lineages from slingshot analysis (N) and cells colored by the inferred slingshot pseudotime (O). (P–R) Selected differentially expressed genes in TRC/BRC/MedRC lineages (P), PRC lineage (Q), and VSMC lineage (R) along the pseudotime. Mesenteric lymph node scRNA-seq data are representative of n = 10−15 mice per time point; 6,029 cells from E18; 8,219 cells from P7; 7,512 cells from 3-wk-old mice; 6,509 cells from 8-wk-old mice. Lymph node anlagen scRNA-seq data are representative of n = 15 embryos from two independent experiments; 2,699 cells in total from Ccl19-iEYFP⁺ embryos and 8,787 cells from Cxcl13-EYFP⁺ embryos.