Fig 1: Prtn3-based fate-tracking reveals early phases of DC development in LNs(A) Expression of the human CD4 reporter among the indicated populations in the BM and LNs of Prtn3CreERT2-hCD4 mice. Black lines show WT controls. CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; GP, granulocyte progenitor; MDP, monocyte-dendritic cell progenitor; cMoP, common monocyte progenitor; Mono, monocytes; Neutro, neutrophils; pDC, plasmacytoid dendritic cell.(B) Diagram showing the labeling/fate-tracking of a wave of myeloid precursors in the BM of Prtn3LSL-Tom mice after tamoxifen injection and the consequent migration to LNs.(C) Frequency of Tomato+ cells among the indicated populations in the BM and LNs of Prtn3LSL-Tom mice 24 h after tamoxifen injection.(D) Frequency of Tomato+ cells among the indicated populations in the BM of Prtn3LSL-Tom mice 1, 3, 5, and 7 days after tamoxifen injection.(E) Frequency of MHCII-, MHCIIlo, and MHCIIhi cells among all (top) or Tomato+ (bottom) cDCs (Lin-(Ly6ChiCD11b+)–B220-CD11c+) in LNs of Prtn3LSL-Tom mice 3, 5, and 7 days after tamoxifen injection.(F–L) Single-cell RNA sequencing (scRNA-seq) analysis of preDCs in LNs.(F) Experimental setup. PreDCs (CD11c+MHCII-CD135+) from LNs of unmanipulated WT mice and Tomato+ preDCs from LNs of Prtn3LSL-Tom mice that received tamoxifen injection 3, 5, or 7 days earlier were sorted and analyzed by scRNA-seq.(G) Uniform manifold approximation and projection (UMAP) plot displaying 8,516 scRNA-seq transcriptomes clustered in 10 different clusters. Lines indicate different lineage trajectories calculated by the Slingshot algorithm starting from cluster 5.(H) UMAP plots displaying expression of Cd24a, Cx3cr1, Sirpa, and Ly6d genes.(I) Dotplot of representative marker genes associated with the identified clusters. Color indicates the Z score mean of the expression values across clusters and dot size represents fraction of cells in the cluster expressing the respective genes.(J) UMAP plot showing the distribution of Tomato+ preDCs among the clusters 3, 5, or 7 days after tamoxifen injection.(K) Normalized percentages of Tomato+ cells within the cDC1 lineage associated clusters (5, 2, and 9) 3, 5, or 7 days after tamoxifen injections.(L) Gene ontology terms enriched among genes that are upregulated during the early development of the cDC1 lineage in LNs. FDR, false discovery rate.Data display one representative of =2 independent experiments (A–D), pooled data from =2 independent experiments (E) or data from 1 experiment (F–L) (A, C, and D, n = 4; E, n = 6). Error bars indicate the mean ± SEM.