Fig. 3. Lymph node anlagen develop normally in Prox1-heterozygous and Prox1-null mouse embryos. To determine whether PROX1 deficiency affects LN development in a dose-dependent manner, we compared the LN anlagen of E14.5 wild-type, Prox1^+/- and Prox1^-/- embryos. (A-C) Sections were stained for CD45 (blue), CD4 (green) and IL7R (red) to identify clusters of LTi cells, which form the LN anlagen. (D-I) Adjacent sections were stained for (D-F) VEGFR1 (green), MECA32 (red) and VEGFR2 (blue) to detect endothelial cells, and for PROX1 (G,H, green), β-galactosidase (β-gal) (I, green), podoplanin (G-I, red) and MAdCAM1 (G-I, blue) to detect lymphatic endothelium and stromal cells within the LN anlagen. LN anlagen of wild-type and Prox1^+/- embryos appeared indistinguishable, although the organization of the lymphatic epithelium and blood endothelium within the LN anlagen was disorganized in Prox1^-/- embryos. Arrows in D and E indicate blood vessels, and arrowheads indicate small blood vessels. Arrows in G and H indicate the lining of the lymphatic endothelium. Scale bars: 75 µm in A-I.