Fig 2: Reticular cell subset differentiation during splenic white pulp development. a, b Time course analysis of EYFP-expressing cells in spleens from Ccl19-iEYFP mice harvested at the indicated stages. Confocal microscopic analysis depicting αSMA+ myofibroblasts (a) and PDPN+ TRC (b). Boxes in (b) indicate magnified regions in the lower row. Microscopy data are representative for two or more independent experiments (n ≥ 4 mice per group). c, d Flow cytometric analysis of CD45−TER119−EYFP+ cells using antibodies against PDPN, CD157 and Sca-1. Values from one representative experiment indicate percentage of the respective population shown in (c) and cumulated values for proportions of reticular cell subsets shown in (d) (n > 5 mice per group from two to three independent experiments, mean ± SEM). e, f Assessment of cellular proliferation using Ki67 staining in spleens of Ccl19-iEYFP mice at the indicated age with representative section in (e) and quantification of the proportion of Ki67 expression in EYFP+ cells (f) (n > 4 mice per group from three independent experiments, mean ± SEM). Statistical analysis was performed using a one-way ANOVA with Tukey’s post test. Source data are provided as Source Data file

Fig 3: Staggered commitment of splenic white pulp organizer cells along the developing arterial tree. a, b Ccl19-iEYFP mice were treated with doxycycline from E19.5 (a, c) or P7 (b, d). Distribution and phenotype of EYFP+ cells in the white pulp of 6-week-old mice were determined by confocal microscopy using the indicated antibodies. Boxes show region of higher magnification in right images, arrows indicate EYFP+MAdCAM-1+ MRC c, d, arrowheads indicate EYFP+PDPN+ TRC (c, d), asterisks indicate EYFP+CD21/35+ FDC (c, d) (scale bars = 500/20 µm). e Determination of EYFP+ cell distances from the central artery at the hilum. Cross-sections from Ccl19-iEYFP spleens from 6-week-old mice were stained with antibody against TER119 and EYFP expression was pseudocolor-coded (lower image) according to concentric distance from the hilum (0–2400 µm). Scale bar = 250 µm. f Frequency of EYFP+ cells in white pulp areas of indicated concentric distances from the central artery (n = 5–7 mice per group from two independent experiments, mean ± SEM). Statistical analysis was performed using a Kolmogorov–Smirnov test. Source data are provided as Source Data file