VEGF receptor heterodimers I Nilsson et al Figure 5 Heterodimers in angiogenic sprouts. (A) Angiogenic sprouting in response to VEGFA or VEGFC. EBs were cultured in 3D collagen matrix in the presence of VEGFA or VEGFC. Microphotographs were taken at day 18 on whole-mount ﬁxed, CD31-immunostained samples. Scale bar ¼ 300 mm. (B) Quantiﬁcation of total vascular area from the data in (A) based on ﬁve EBs per condition and expressed as fold induction ±s.d. (C) Expression of VEGFR2 and VEGFR3 in angiogenic sprouts. Immunostaining for VEGFR2 (green; upper panels) and VEGFR3 (green; lower panels) on CD31-positive (red) angiogenic sprouts in 3D EBs treated with VEGFA or VEGFC. The orientation of the tip versus stalk is indicated. Scale bar ¼ 10 mm. (D) Quantiﬁcation of VEGFR2 and VEGFR3 expression in the tip cell region compared with entire angiogenic sprouts. n ¼ 4. (E) Location of heterodimers on VEGFC-induced angiogenic sprouts. Red spots represent PLA reactions in tip cells. Panels show PLA spots in CD31-positive angiogenic tip cell regions. Lower panels show saturation of CD31-positivity to better visualize the ﬁlopodia. Note that PLA spots are located on ﬁlopodia extending ahead of the tip cell. Scale bar ¼ 10 mm. (F) Quantiﬁcation of in situ PLA detecting VEGFR2/-3 heterodimers in angiogenic sprouts in response to VEGFA or VEGFC in 3D EB cultures. n ¼ 8. (G) Quantiﬁcation of heterodimers in tip and stalk cells. VEGFA-treated angiogenic sprouts contained heterodimers evenly distributed over the sprouts, whereas VEGFC-treated angiogenic sprouts showed accumulation of heterodimers in tip cells. n ¼ 8. Asterisks in panels B, D, F–G indicate degree of signiﬁcance (*Po0.05, **Po0.01, ***Po0.001). effect of the VEGFR3-neutralizing antibodies is to block formation of VEGFR2/-3 heterodimers. We thus conclude that VEGFR2/-3 heterodimerization in this context serves to positively regulate sprouting angiogenesis. Discussion Molecular interactions such as dimerization of receptor tyr- osine kinases are vital in cellular communication. We show herein that in situ PLA can successfully be used to detect native VEGFR2 and VEGFR3 homodimers as well as VEGFR2/-3 heterodimers. Of note, detection of such native complexes cannot be done using co-immunoprecipitation methodology. In contrast to our recent in situ PLA study on & 2010 European Molecular Biology Organization epidermal growth factor (EGF) receptor/Her2 homo- and heterodimerization, which occurs in the absence of ligand (Leuchowius et al, 2009), formation of VEGFR complexes was strictly ligand dependent. Using in situ PLA, we detected about 3–4-fold more heterocomplexes/cell in response to VEGFC as compared with VEGFA. The fold-induction of heterocomplexes compared with basal levels in unstimulated cells was about a 100-fold for VEGFC. The speciﬁcity of the methodology was validated (1) by the use of neutralizing anti-receptor antibodies, which block the ligand-binding site on the receptors; (2) by the use of an irrelevant ligand (PDGFB); and ﬁnally (3) by performing kinetic analyses that showed disappearance of the PLA spots with time, in conjunction with ligand-induced internalization of receptors. The EMBO Journal VOL 29 | NO 8 | 2010 1383