Molecular characterization of Ccl19-tTA ⁺ and Cxcl13-Cre ⁺ FRC and VSMC progenitors in inguinal and mesenteric lymph node anlagen. (A) Schematic representation for the analysis of inguinal and mesenteric lymph node anlagen from Ccl19-iEYFP⁺ and Cxcl13-EYFP⁺ embryos at E18. (B and C) Whole-mount confocal microscopy analysis of inguinal (B) and mesenteric (C) lymph node anlagen from Ccl19-iEYFP embryos at E18. Arrows highlight the localization of Ccl19-iEYFP⁺ cells inside the lymph node anlage. Microscopy images are representative for three inguinal and three mesenteric lymph node anlagen from three independent experiments. Scale bars: 100 µm (B) and 200 µm (C). (D and E) Whole-mount confocal microscopy images showing inguinal (D) and mesenteric (E) lymph node anlagen from Cxcl13-EYFP⁺ embryos at E18. Arrows highlight the localization of Cxcl13-EYFP⁺ cells in the lymph node anlage. Arrowhead highlights the appearance of Cxcl13⁺ cells in the mesenchyme around the lymph node anlage. Microscopy images are representative for three inguinal and three mesenteric lymph node anlagen from three independent experiments. Scale bars: 100 µm (D) and 200 µm (E). (F) scRNA-seq analysis of Ccl19-iEYFP⁺ cells from inguinal and mesenteric lymph node anlagen at E18. UMAP representation of Ccl19-iEYFP⁺ cell clusters colored by lymph node entity (left panel) and Ccl19 expression (right panel). (G) scRNA-seq analysis of Cxcl13-EYFP⁺ cells from inguinal and mesenteric lymph node anlagen at E18. UMAP representation of Cxcl13-EYFP⁺ cell clusters colored by lymph node entity (left panel) and Ccl19 expression (right panel). (H) scRNA-seq analysis of Cxcl13-EYFP⁺ cells filtered for Ccl19 expression colored by cluster identity. (I–K) UMAP representation of Cxcl13-EYFP⁺Ccl19⁺ cells and projection of genes associated with cell proliferation (I), perivascular/mural (J), and FRC (K) signatures on the scRNA-seq dataset. (L–S) scRNA-seq analysis of Ccl19-iEYFP⁺ cells isolated from inguinal and mesenteric lymph node anlagen at E18. (L) Dot plot indicating the average expression of signature genes across embryonic Ccl19-iEYFP⁺ cell populations. (M) UMAP representation of Ccl19-iEYFP⁺ cell clusters. (N–P) Projection of proliferation (N), perivascular/mural (O), and FRC (P) signatures consisting of the indicated genes on the scRNA-seq dataset. (Q and R) Projection of selected genes associated with stem cell proliferation (Q) and stem cell population maintenance (R) on the scRNA-seq dataset. (S) Differentiation trajectory analysis of Ccl19-iEYFP⁺ cells from mesenteric lymph nodes using slingshot colored by the inferred slingshot pseudotime. 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.