Fig. 4: Impaired developmental lymphangiogenesis upon efficient deletion of Vegfr2 in LECs.

Genetic constructs (a) and experimental scheme (b) for efficient deletion and faithful tracing of Vegfr2 or double Vegfr2/Vegfr3-deleted LECs using R26-iSuRe-HadCre. c, d Whole mount immunofluorescence showing the distribution of MbTom⁺ LECs, labelled at early postnatal development (P1, P2), within the mature dermal vasculature in 3-week-old mice. Cre⁺ mice carrying only R26-iSuRe-HadCre (+/+) or heterozygous (flox/+) Vegfr2 allele show contribution of MbTom⁺ LECs in both lymphatic capillaries and collecting vessels, while Vegfr2-deleted (flox/flox) mice show labelling predominantly in collecting vessels. Boxed areas in (c) are magnified in (d) to show limited contribution of recombined cell to lymphatic capillaries and efficient deletion of VEGFR2 in Tom⁺ collecting lymphatic vessels in Vegfr2^flox/flox mice. In (d), dotted lines in VEGFR2 staining indicate the outline of collecting vessels, and arrowheads point to non-recombined LECs in mutant mice. Images in (c, d) represent data from two independent experiments. Whole mount immunofluorescence (e and magnification of boxed areas in (f)) and quantification of vessel area (g), branchpoints (h) and terminal lymphatic capillary ends (i), showing that continuous deletion of Vegfr2 results in hypoplastic lymphatic network in Vegfr2-deleted (flox/flox) mice compared to controls. Arrowheads in (f) point to non-recombined LECs at vessel tips in mutant mice. Data in (g–i) represent mean ± s.d. (n = 3–4 mice per genotype as indicated). *p < 0.05, ****p < 0.0001; One-way ANOVA followed by Tukey’s multiple comparison test. j Whole mount immunofluorescence of P21 ear dermis showing the effect of neonatal high-fidelity deletion of Vegfr2 and/or Vegfr3. Images in (j) represent data from two independent experiments. Scale bar: 500 µm (c, e, f, j), 200 µm (d).