Fig. 3: Reduced contribution of VEGFR2 signalling-defective LECs to developing dermal lymphatic capillaries.

Genetic constructs (a) and experimental scheme (b) for tracking of Vegfr2-deleted LECs using the iSuRe-Cre allele that switches upon conditional CreER^T2 activation to expression of constitutive Cre and MbTomato. c, d Whole mount immunofluorescence showing distribution of MbTom⁺ LECs, labelled at early postnatal development, within mature dermal vasculature in 3-week-old mice (c). Cre⁺ mice carrying a heterozygous (flox/+) Vegfr2 allele show contribution of MbTom⁺ LECs in both lymphatic capillaries (arrowhead) and (pre-)collecting vessels (arrow), Vegfr2-deleted (flox/flox) mice show labelling predominantly in (pre-)collecting vessels (arrow). Images (c, d) show representative data from 3 independent experiments. Boxed areas are magnified in (d), showing efficient VEGFR2 depletion in Vegfr2-deleted (flox/flox) mice. Dotted lines in VEGFR2 and LYVE1 staining in (d) indicate the outline of collecting vessels. Arrowheads in (d) indicate lymphatic valves. Flow cytometry analysis of LEC subtypes (e) and MbTom⁺ LECs (f) in P21 ear skin. Data represent mean ± s.d. (n = 5-10 mice per genotype as indicated). ***p < 0.001; Two-sided Mann-Whitney U test. g Genetic constructs and experimental scheme for tracing ECs with different levels of VEGFR2 activity after inducing mosaic expression of constitutively active VEGFR2 (tdTom-Vegfr2^ac) or a dominant negative tyrosine kinase mutant form (Y1173) of VEGFR2 coupled to EYFP expression (EYFP-Vegfr2^TK-). mKate2 expression is not coupled to VEGFR2 expression and is not visualized. h Whole mount immunofluorescence staining of P21 ear dermis of R26-iMb-Vegfr2;Cdh5-CreER^T2 mice showing abundant presence of LECs expressing tdTom-Vegfr2^ac in lymphatic capillaries, while LECs expressing EYFP-Vegfr2^TK- are only found within collecting vessels. VEGFR3⁺ vessels are pseudo-coloured in light blue in single channel images. Red arrows indicate LECs expressing tdTom-Vegfr2^ac and green arrow LECs expressing EYFP-Vegfr2^TK-. i Contribution of EYFP-Vegfr2^TK- and tdTom-Vegfr2^ac LECs to dermal lymphatic vasculature, shown as the proportion of vessel segments between two valves within LYVE1⁺ capillary^- (Cap), or collecting vessels (Col), with reporter-positive cells. Data represent mean ± s.d. (n = 450 (Cap, 5 mice) and n = 636 (Col, 5 mice) vessel segments; each dot represents data from one ear). ****p < 0.0001; Fisher’s exact (two-tailed). Scale bar: 250 µm (c), 200 µm (h), 50 µm (d).