Fig 2: YAP/TAZ support growth and structural organization of LNs by FRCs.a Diagram for generation of indicated mice and their analyses at 8 weeks after birth. b, c Representative images of YAP or TAZ in PDGFRß+ or CCL19+ FRCs in WT and Yap/Taz?FRC mice. FRCs around high endothelial venule (HEV) within the white dashed-line box are magnified in the lower panels with single-channel YAP or TAZ image. Scale bars, 250 µm. d Comparisons of body weight, inguinal LN weight and total number of cells within the inguinal LN in WT (n = 11; body weight) and Yap/Taz?FRC mice (n = 9; body weight). e Representative flow cytometric analysis and comparison of proportion of PDPN+CD31-FRCs (red box) gated from CD45- stromal cells of skin-draining LNs in WT and Yap/Taz?FRC mice. f Representative images and comparison of Ki-67+ FRCs (white arrows) in WT and Yap/Taz?FRC mice. Scale bars, 50 µm. g Comparison of indicated stromal cell counts gated from CD45- cells of skin-draining LNs in WT and Yap/Taz?FRC mice. BECs (n = 5), blood endothelial cells; LECs (n = 6), lymphatic endothelial cells. h Representative images of distinction between B and T cells (white dashed line) beneath the LN capsule (white line) in WT and Yap/Taz?FRC mice. Scale bars, 200 µm. i Comparison of indicated mRNA expression in FRCs sorted from WT and Yap/Taz?FRC mice (quintuplicate values using n = 10–15 mice/group). j, k Representative images and comparison of DsRed+ B cells and GFP+ T cells within the inguinal LN at 24 h after the adoptive transfer in WT and Yap/Taz?FRC mice. Scale bars, 500 µm. l Changes in body weight after 1 × 103 pfu of A/PR/8 influenza viral infection (n = 13). m Flow cytometric analyses and comparisons of IFN-?+CD8+ T cells in gated CD3e+ T cells. n = 5 (CO) or 7 (IM) mice. Unless otherwise denoted, each dot indicates a value obtained from one mouse and n = 4 mice/group pooled from two independent experiments. Horizontal bars indicate mean ± SD and P values versus WT by two-tailed Mann–Whitney U test. NS, not significant.

Fig 3: YAP/TAZ hyperactivation impairs differentiation and maturation of FRCs.a Diagram for analyses of indicated mice at P14. b Representative images of PDGFRß+ FRCs and CD31+ vessels in WT and Lats1/2?FRC mice (n = 5). Scale bars, 500 µm. c Comparisons of LN weight (n = 4–7) and total number of cells (n = 6–10) in WT and Lats1/2?FRC mice. d Diagram for analyses of indicated mice at E18.5 or P14. e Representative images of LN anlagen (dashed line) at E18.5 showing CD4+ LTi cells in WT and Lats1/2?FRC mice (n = 6). Scale bars, 200 µm. f, Representative images of indicated markers (dashed box) within the inguinal LN (dotted-line) in WT and Lats1/2?FRC mice at P14 (n = 6). Scale bars, 500 µm. g, h Diagram and representative images for analyses of WT?FRC-TR mice (n = 6) that were injected with anti-CD3e for 5 days to induce T cell depletion. Scale bars, 100 µm. i Representative flow cytometric plots and comparison of proportion of PDPN+CD31- FRCs (red box) and PDPN-CD31- double-negative (DN) cells of skin-draining LNs in WT and Lats1/2?FRC (n = 5–6) mice. j Representative images and comparison of YAP expression and nuclear localization (green-arrowheads) in LN of WT and Lats1/2?FRC mice (n = 5). Scale bars, 20 µm. k Comparison of indicated mRNA expression in FRCs sorted from WT?FRC-TR and Lats1/2i?FRC-TR mice (n = 4). l Representative images and comparisons of indicated marker expressions in LNs of WT and Lats1/2?FRC mice (n = 4–5). Scale bars, 20 µm. m Comparison of indicated mRNA expression in FRCs sorted from WT?FRC-TR and Lats1/2i?FRC-TR mice. n Diagram for analyses of indicated mice at P14. o Representative images of YAP expression in LNs of WT and L1/2-Y/T?FRCmice. Scale bars, 100 µm. p, q Representative images of indicated markers in LNs of WT and L1/2-Y/T?FRC mice. Scale bars, 500 µm. Unless otherwise denoted, each dot indicates a value obtained from inguinal LN and n = 4 mice. Horizontal bars indicate mean ± SD and P values versus WT or WT?FRC-TR by two-tailed Mann-Whitney U test except for (k) and (m) (two-tailed Student’s t-test). NS, not significant.

Fig 4: Loss of antigen-bearing LECs results in antigen presentation. a Experimental design using anti- PDPN to expand lymphatic endothelial cells. b Wild-type mice were immunized subcutaneously with fluorescent antigen (10 µg/site) along, or in combination with two doses of the anti-PDPN antibody (100 µg iv). One week after injection, mice were killed and the presence of antigen was visualized by flow cytometry. c Antigen+ lymphatic endothelial cells were quantified at 1 and 2 weeks after antigen and PDPN treatment as in a. Mice of each treatment were pooled from two independent experiments with three mice per group per time point, where error bars represent the mean ± standard error of the mean. Statistical analysis was done using two-way ANOVA with a p-value of 0.0123 represented using one asterisk. d OT1 T cells were transferred 3 days before killing at 1 and 2 weeks, and e their percent division was calculated and normalized to 1 in order to visualize differences between weeks rather than due to an effect on T-cell division caused by PDPN treatment. Mice of each treatment were pooled from two independent experiments with three mice per group per time point. Statistical analysis was done using two-way ANOVA with a p-value of 0.0031 represented using two asterisks in e T-cell division in antigen alone (gray shaded) and antigen with PDPN treatment (black line) 2 weeks post injection of antigen. Error bars shown in the figure are mean and standard error of the mean

Fig 5: Cell fate mapping of embryonic lymphoid tissue organizer cells. a Schematic depiction of the experimental approach. Spleens have been harvested from E19.5 to 6 week fate mapped, b Ccl19-iEYFP, and c Ccl19-iEYFP Ltbrfl/fl mice. Cross-sections were stained with the indicated antibodies and analyzed by confocal microscopy. Boxes indicate representative white pulp areas shown at higher magnification in (d–g) (scale bars = 500 µm). d, e Confocal microscopy analysis of fate-mapped Ltbr-proficient EYFP+ white pulp reticular cell subsets highlighting PDPN+ TRC (d), MAdCAM-1+ MRC (d) and CD21/35+ FDC (e), scale bars = 20 µm/5 µm. f, g Fate-mapped Ltbr-deficient EYFP+ cells by confocal microscopy using indicated marker combinations, scale bars = 20/5 µm. Microscopy data are representative for n = 7 mice per group from three independent experiments. h Representative flow cytometric analysis of fate-mapped EYFP+ cells from Ccl19-iEYFP and Ccl19-iEYFP Ltbrfl/fl spleens, gated on and analyzed for the indicated markers. Values indicate percentage of respective populations. i Percentage of EYFP+ cells from spleens of indicated mouse strains, based on gating in (h) (n = 7 mice per group from three independent experiments, mean ± SEM). j, k Assessment of cellular proliferation using Ki67 staining in spleens of Ccl19-iEYFP and Ccl19-iEYFP Ltbrfl/fl mice at the indicated age with representative section in (j) and quantification of the proportion of Ki67 expression in EYFP+ cells in (k) (n = 3–5 mice per group from three independent experiments, mean ± SEM). Statistical analyses were performed using a Mann–Whitney test. Source data are provided as Source Data file