DEV028456 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Lymph node (LN) anlagen contain RORγt-expressing lymphoid tissue inducer (LTi) cells. To confirm that the clustered CD45⁺/CD4⁺ cells that colocalize with MAdCAM1⁺ cells are indeed LTi cells, we immunostained sections of (A) E16.5 wild-type, (B) E14.5 wild-type and (C) E14.5 Prox1^−/− embryos with antibodies against RORγt (green), CD4 (red) and CD45 (blue). RORγt expression was detected in CD45⁺/CD4⁺ cells of wild-type and Prox1-null embryos, confirming their identity as LTi cells (arrowheads).

• Supplemental Figure S2 -

  Fig. S2. The overall architecture of adult lymph nodes from Prox1-heterozygous mice appears indistinguishable from that of wild-type mice. Peripheral LNs from wild-type or Prox1-heterozygous mice were collected and stained for CD4 (green) and CD8 (red) to identify T-cell subsets, and B220 (blue) to detect B cells (A,B). Follicular dendritic cells within the B-cell follicle were identified by expression of CD35 (red) and CD16/32 (green), and fibroblastic reticular cells within the T-cell areas were characterized by expression of podoplanin (blue) (C,D). High endothelial venules within the T-cell areas were identified by expression of CD105 (red) and PNAd, as detected by staining for MECA-79 (green), and were surrounded by LYVE1⁺ cells (blue) (E,F). Podoplanin⁺ (blue) fibroblastic reticular cells within the T-cell areas colocalized with ERTR7⁺ (green) extracellular matrix components, and some fibroblastic reticular cells expressed PROX1 (red) (G,H).