Fig. 7: Expression, cell surface localization and activation of VEGFR2 and VEGFR3 and their heterodimers in lymphatic endothelium.

a ScRNA-seq bubble plots showing expression of genes encoding the VEGF/VEGF-C receptors (Vegfr1/Flt1, Vegfr2/Kdr, Vegfr3/Flt4) and co-receptors (Nrp1, Nrp2) in dermal LEC types from mouse ear skin. Data extracted from¹⁸. Col, Collecting vessel; (Ptx3) Cap, (terminal Ptx3⁺) lymphatic capillary. Whole mount immunofluorescence of 4-week-old mouse ear dermis (b), and E15 embryonic back skin (c) for total (permeabilized) or cell surface (unpermeabilised) levels of VEGFR2 and VEGFR3. Boxed regions in (c) are magnified on the right. Relative LEC:BEC mean VEGFR2 fluorescence staining intensity is indicated. Images represent data from two (b) or a single (c) experiment(s). Whole mount immunofluorescence of the ear skin showing the effect of VEGF-C (d, e) and Prox1-CreER^T2-mediated deletion of Pik3ca (e) on VEGFR2 and VEGFR3 levels and localization. Recombination was induced at 3 weeks by five consecutive administrations of tamoxifen (1 mg). Two weeks later, AAV9-VEGF-C was intradermally injected into the ear skin and mice (n = 2–4 per condition) were analyzed two weeks later. Ctrl, control group including Cre- mice and Cre+ not carrying the floxed allele (see Source Data). Images in (d) represent data from two independent experiments. f Quantification of VEGFR2 levels from (e), represented as corrected total cell fluorescence (CTCF) normalized to control values. Data are shown as a superplot: round symbols represent single measurements, triangles indicate mean value per mouse (n = 4 (Cre wt ± VEGF-C, permeabilized); n = 3 (flox/flox ± VEGF-C, permeabilized); n = 3 (Cre wt ± VEGF-C, unpermeabilized); n = 2 (flox/flox ± VEGF-C, unpermeabilized) mice, 20-30 measurements per mouse). Statistical analyses were performed on mouse means and shown as mean ± s.d. ***p < 0.001; One-way ANOVA followed by Tukey’s multiple comparison test. g Whole mount PLA of activated VEGFR2 or VEGFR3 in E15 skin, detected using anti-phospho-Tyrosine and anti-VEGFR antibodies, co-stained with PECAM1 or LYVE1. (h) Whole mount PLA for the detection of VEGFR2 and VEGFR3 heterodimers in E15 skin. Images in (g, h) represent data from 3 independent experiments. Boxed regions in (g, h) are magnified on the right. Scale bar: 250 µm (b, c), 50 µm (d, e, g, h).