VEGF receptor heterodimers I Nilsson et al Figure 4 Formation of vessel structures in differentiating 2D EBs involves both VEGFR2 and VEGFR3. (A) Expression of VEGFRs in 2D EB vascular structures. EBs differentiating in 2D cultures for 12 days in the presence of VEGFA shows vessel-like structures co-expressing VEGFR2 and VEGFR3 (upper panels; merged immunostainings to the right), or VE-cadherin and VEGFR3 (lower panels; merged immunostainings to the right). Scale bar ¼ 50 mm (upper), 100 mm (lower). (B) Complex formation between VEGFRs in 2D EBs. Left: Immunoprecipitation (ip) of VEGFR3 from day 12 EBs treated with vehicle (–) VEGFA (A) or VEGFC (C) for 15 min followed by immunoblotting for VEGFR2 (upper left panel). Immunoblotting for VEGFR3 (lower left panel) shows equal loading of VEGFR3. Right: Parallel aliquots of cell lysate were analysed by immunoprecipitation of VEGFR2 followed by immunoblotting for VEGFR3 (upper right panel). VEGFR2 immunoblotting (lower right panel) showed equal loading. (C) Heterodimers in CD31-positive cells. Formation of VEGFR2/-3 heterodimers as detected by in situ PLA (red spots) on 2D EBs immunostained for CD31 (green), in response to vehicle (–), VEGFA or VEGFC. Scale bar ¼ 10 mm. (D) Quantiﬁcation of PLA spots in CD31-positive cells as in C. n ¼ 8. (E) Identiﬁcation of LYVE1-positive cells. EBs in 2D cultures were treated with VEGFA or VEGFC until day 12, and immunostained to detect expression of CD31 (green) and LYVE1 (red). Panels to the right show merged immunostainings. Lymphatic vascular structures expressing LYVE1 but not CD31 are indicated by arrows in the VEGFC-treated cultures. Single, rather than vessel-organized LYVE1-positive cells, are indicated by asterisk. Scale bar ¼ 100 mm. (F) Heterodimers in LYVE1-positive cells. EBs in 2D culture were treated as indicated above and processed for immunostaining to detect CD31 (blue) and LYVE1 (white), followed by in situ PLA to detect VEGFR2/-3 heterodimers (red). Scale bar ¼ 10 mm. (G) Quantiﬁcation of PLA spots in LYVE1-positive cells as in (F). n ¼ 8. Asterisks in panels D and G indicate the degree of signiﬁcance (**Po0.01, ***Po0.001). stages (Olsson et al, 2006). In accordance, we have shown earlier that VEGFR2 neutralization attenuates EB vasculariza- tion (Magnusson et al, 2004). The contribution of VEGFR2 to the different stages of endothelial cell development can there- fore not readily be assessed by this approach. Figure 6A and B shows that VEGFA-induced angiogenic sprouting was signiﬁcantly decreased by treatment with the VEGFR3-neutralizing antibody but not by a control IgG antibody. As shown in Figure 6C, the VEGFR3 neutralizing antibody did not block VEGFA-induced VEGFR2 phosphory- lation. The transient increase in phosphorylated VEGFR2 level at the 2 min time point may be due to increased VEGFR2 homodimerization and more efﬁcient phosphoryla- tion under conditions when VEGFR3 is neutralized (compare VEGFR2 tyrosine phosphorylation in response to VEGFA and VEGFC in Figure 1B, middle panel). As VEGFA induces VEGFR2 homodimers and VEGFR2/-3 heterodimers but not VEGFR3 homodimers (Figure 3), these data indicate that the 1382 The EMBO Journal VOL 29 | NO 8 | 2010 & 2010 European Molecular Biology Organization