Figure 3.

Different proliferation dynamics of LECs and BECs during Pik3ca-driven vascular overgrowth. (A–D) Whole-mount immunofluorescence of ear skin analyzed at different stages after 4-OHT administration, and quantification of S-phase cells (EdU⁺) and all cycling cells (Ki67⁺; arrowheads) in Pik3ca^H1047R;Vegfr3-CreER^T2 (A and B) and Pik3ca^H1047R;Vegfr1-CreER^T2 (C and D) mice. EdU was administered 16 h prior to analysis. LYVE1 and PROX1 were used for the identification of LECs, and EMCN for the identification of (venous) BECs. IMARIS surface mask based on LYVE1 expression was used to extract LEC-specific Ki67/EdU signals. The original unmasked images are shown in Fig. S2 F. Note initial proliferative response in both models, but sustained proliferation only in the LECs of Vegfr3-CreER^T2 mice. (E) Flow cytometry analysis of proliferating dermal LECs and BECs in Pik3ca^H1047R;Vegfr3-CreER^T2 and Pik3ca^H1047R;Vegfr1-CreER^T2 mice, respectively, and their littermate controls at the indicated stages. Data represent mean % of Ki67⁺ ECs (n = 3–7 mice) ± SEM. (F) Proportion of LECs and BECs out of all ECs in the Pik3ca^H1047R;Vegfr3-CreER^T2 and Pik3ca^H1047R;Vegfr1-CreER^T2 animals analyzed in E. P value in B and D–F: two-tailed unpaired Student’s t test; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; ns, P > 0.05. Scale bar: 50 μm (A and C).