Extended Data Fig. 5: TRCs undergo distributed clonal expansion.
From: Multitier mechanics control stromal adaptations in the swelling lymph node
(a) Schematic of the mosaic analysis with double markers (MADM) labeling principle. Rare interchromosomal recombination in the G2 cell cycle phase following x-segregation of chromosomes labels T-zone reticular cells (TRCs) with either a cytoplasmic tdTomato or GFP. (b) Schematic of the sparse mosaic analysis with double markers (MADM) labeling approach for TRC cluster analysis in popliteal lymph nodes (LNs) from Ccl19-Cre MADM-7GT/TG mice in homeostasis (day 0) and inflammation (day 4 and 8) (left). Sparse labeling of TRCs in a histological section of a homeostatic LN (right). Scale bar = 200 µm. (c) 3D fluorescent intensity cropped images from light-sheet fluorescent microscopy of entire lymph nodes for which MADM-GFP labeled TRCs and in situ labeled high endothelial venules (HEVs) by PNAd-ATTO647n Ab are shown, as in b. Scale bars = 20 µm. (d) Example of the labeling of HEVs and mapping of MADM-labeled TRCs (only tdTomato+ TRCs are shown) at inflammation (day 4) as in c. The enlarged image depicts the mapping of individual TRCs by a grey sphere at the center of each cell. Scale bars = 200 µm. (e) Quantification of number of MADM-labeled TRCs as in c (n = 5, 5, 5). (f) Quantification of number of MADM-labeled TRCs found in clusters as in c (n = 5, 5, 5). (g) Quantification of numbers of MADM-labeled TRCs found in cluster as percentage of total MADM-labeled TRCs as in c (n = 5, 5, 5). (h) Quantification of the average distance to the nearest neighbor (NN) of both observed and simulated TRCs as in c (n = 5, 5, 5, 5, 5, 5). Data from d, f, g shown as mean ± s.d. and h shown as mean. Datapoints in e, f, g, h represent independently analyzed LNs. Statistical analysis was performed using Kruskal-Wallis test (e, f, g) and paired two-tailed t test. All experiments were repeated independently (≥3 mice and ≥2 experiments). For statistical details see Supplementary Information, table 1. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001.
