Lower expression level of Tie2 in LECs. (A) FACS analysis of the distribution of LYVE-1⁺ cells in embryogenesis. E11, E13, and E15 mouse embryos were dissected. Embryonic liver and spleen tissues were excluded microscopically, and other tissues were treated with collagenase and Dispase and used as a source of material for FACS analysis. These cells were double-stained with ALY7 and CD31/PECAM-1 mAb (upper panels), CD34 mAb (middle panels), and anti-Tie2 mAb (lower panels). Box in each panel indicates LYVE-1⁺ LEC fraction, respectively. (B) Expression of CD34 and LYVE-1 in CD45^-CD31⁺ cells. mRNA was isolated from LYVE-1⁺ CD34^low/- as LECs (R1) or LYVE-1^- CD34⁺ as BECs (R2) from E14 embryos in which liver and spleen tissues were excluded. (C-D) RT-PCR analysis of genes served as markers for LECs/BECs. mRNA from nonsorted cells without reverse-transcriptase treatment was used as a negative control (RT(-)). (C) Note that expression of Prox1, podoplanin, and secondary lymphoid chemokine (SLC) was restricted to R1, and VEGFR-3 expression was very high in R1, whereas CD31/PECAM-1 was expressed equally in both R1 and R2. Tie2 expression in R1 was weaker than that seen in R2. (D) Note that expression of neuropilin-1 (Nrp-1), Flt-1, and CD44 was very high in R2, but faint in R1. In contrast, the transcript of both Flk-1 and Tie1 was equally expressed in R1 and R2, respectively. Expression of GAPDH was used as an internal control.