potent in this regard. VEGFR2 homodimers were induced only by VEGFA and not by VEGFC (Figure 3C). VEGFR3 homodimers were induced by VEGFC (Figure 3D). There was a slight tendency for VEGFA-induced VEGFR3 homodi- merization, but still the effect was negligible compared with that of VEGFC. Figure 3E concludes on these results; VEGFA induces VEGFR2 homodimers and VEGFR2/-3 heterodimers. VEGFC induces VEGFR3 homodimers and VEGFR2/-3 hetero- dimers. Although no ﬁrm conclusions can be made as reasoned above, homodimers of VEGFR3 seemed to be roughly two-fold more frequent than heterodimers in VEGFC-treated cells. Moreover, homodimers of VEGFR2 were eight-fold more frequent than heterodimers in VEGFA- treated cells. VEGFR2/-3 heterodimers are formed in blood and lymphatic endothelium in differentiating EBs To identify the biological signiﬁcance of VEGF receptor heterodimerization, we exploited the embryoid body (EB) model of angiogenesis (Jakobsson et al, 2007). In this model system, differentiating mouse embryonic stem (ES) cells efﬁciently form capillary structures in response to angiogenic growth factors. In the ﬁrst set of experiments, EBs were cultured in the so-called two-dimensional (2D) setup, seeded onto tissue culture slides. The advantages of the 2D setup are that these cultures readily can be scaled up to allow for biochemical analyses, such as immunoprecipitation, and that the 2D setup also makes it feasible to analyse the relatively rare events whereby immature capillaries in EB cultures begin to transdifferentiate into early lymphatic struc- tures (Kreuger et al, 2006). Figure 4A shows co-expression of VEGFR2 and VEGFR3 in endothelial cells in vascular structures formed in 2D EBs differentiated for 12 days, when endothelial cells constitute about 5% of the total cellular pool (Magnusson et al, 2004). The vascular identity of the cells co-expressing VEGFR2 and VEGFR3 was validated by positive immunostaining for VE- cadherin (lower panel in Figure 4A). As shown in Figure 4B, left panel, immunoprecipitation/immunoblotting analyses demonstrated VEGFC-induced VEGFR2/-3 heterodimeriza- tion in the EB model. Parallel VEGFR2 immunoprecipitation from VEGFA or VEGFC-treated EBs followed by immuno- blotting for VEGFR3 (Figure 4B, right panel) showed VEGFC-, but also to some extent, VEGFA-induced receptor heterodimerization. We next used in situ PLA to examine VEGFR2/-3 hetero- dimer formation in intact 2D EBs. As shown in Figure 4C (quantiﬁcation in Figure 4D), VEGFA and VEGFC induced heterodimers to an extent similar to that detected in the HSaVEC cultures (see Figure 2 for comparison). Occasional PLA signals were detected also in cells expressing low or non- detectable levels of CD31, prompting us to ask whether some of these cells could be LECs or progenitors thereof. We have shown earlier that differentiation of ES cells allows formation of lymphatic endothelial precursors that express lower levels of CD31, which can be identiﬁed by virtue of expression of lymphatic markers such as LYVE1 (Kreuger et al, 2006). As shown in Figure 4E, VEGFA-treated EB cultures contained LYVE1-positive cells that also expressed CD31. A certain fraction of the LYVE1-expressing cells lacked detectable CD31 expression (arrow in Figure 4E). LYVE1-positive cells with distinct morphology, possibly corresponding to & 2010 European Molecular Biology Organization VEGF receptor heterodimers I Nilsson et al monocytic CD45-positive cells, were also detected (asterisk in Figure 4E; see Kreuger et al, 2006). LYVE1-positive cells contained VEGFR2/-3 heterodimers, particularly in response to VEGFC, as shown using in situ PLA (Figure 4F; quantiﬁca- tion in Figure 4G). The number of PLA signals/cell was lower in this analysis; this is in part likely due to that co-staining for LYVE1/CD31 together with PLA required additional washing steps and therefore, loss in sensitivity. However, the relation- ship between the different conditions (control, VEGFA, VEGFC) remained the same. Tip cell ﬁlopodia assemble VEGFR2/-3 heterodimers in response to VEGFA or VEGFC Next, the function of VEGFR2/-3 heterodimers in angiogenic sprouting was addressed. For this purpose, EBs were placed in a three-dimensional (3D) collagen I matrix that is permis- sive for the formation of high-quality angiogenic sprouts, shown to develop a complete basement membrane, to be luminized and covered by pericytes (Jakobsson et al, 2007; Jakobsson and Claesson-Welsh, 2008). As shown in Figure 5A, the 3D EB cultures treated with VEGFA displayed numerous sprouts extending from the core of the EB into the collagen I gel. Fewer and shorter sprouts were formed in cultures receiving VEGFC (see quantiﬁcation in Figure 5B). Angiogenic sprouts were wrapped in a pericyte coat of NG2- positive cells both in VEGFA and VEGFC-treated cultures (Supplementary Figure S1). VEGFR2 expression was detected in CD31-positive en- dothelial cells throughout the angiogenic sprout to a similar extent in response to VEGFA and VEGFC (Figure 5C; quanti- ﬁcation in Figure 5D). There was a tendency for reduced CD31 expression in the tip region of the sprout. VEGFR3 expression in CD31-positive cells was detected in both VEGFA and VEGFC-induced sprouts (Figure 5C). Interestingly, in the VEGFC-treated cultures, VEGFR3 expression was elevated in the tip region compared with the stalk region of the sprout (Figure 5D). In situ PLA signals representing VEGFR2/-3 heterodimerization were detected in the 3D EB sprouts (Figure 5E; see quantiﬁcation in Figure 5F). There was a low background in the untreated controls, and a similar relative degree of VEGFA- and VEGFC-induced heterodimer- ization as seen in monolayer cultures of HSaVECs and in 2D EBs. The heterodimer complexes were localized along the angiogenic sprouts both on the cell bodies and on the end of ﬁlopodia extending from the stalk and tip cells (Figure 5E, left). Note that the in situ PLA signals tended to coalesce into larger ﬂuorescent clusters, indicating that the heterodimers were not evenly distributed over the plasma membrane. Heterodimers were more concentrated in the tip cells relative to the stalk cells (Figure 5E, right) in response to VEGFC, whereas VEGFA-treated sprouts displayed a more even heterodimer distribution (Figure 5G). VEGFR3 blockade prevents heterodimer formation leading to reduced angiogenesis To study the function of heterodimerization in angiogenic sprouting, 3D EB cultures were treated with VEGFA in the presence and absence of neutralizing antibodies against mouse VEGFR3 (Pytowski et al, 2005) (Figure 6A; see quan- tiﬁcation in Figure 6B). We avoided treating cultures with VEGFR2 neutralizing antibodies, as VEGFR2 is strictly re- quired for differentiation of endothelial cells from precursor The EMBO Journal VOL 29 | NO 8 | 2010 1381