a–c, Investigation of lymphatic growth in the trachea of E16.5 wild-type (denoted as Prox1) and Prox1∆CPT1A embryos. a, LYVE1+ lymphatic structures in the trachea. Scale bars, 200 μm. Bottom panels, high magnification images of regions denoted by the white boxes. In the trachea, lymphatic vessels first grow horizontally, after which sprouts branch off vertically (white arrows). Note the underdevelopment of lymphatic growth in the mutant embryo (yellow arrowheads). b, c, Quantitation of the average length of vertical connections (b) and average number of branch points (c) (n = 6 embryos for wild type; n = 5 embryos for Prox1∆CPT1A; 2 litters). d–i, Experiments in E16.5 wild-type control (denoted as Flt4) and Flt4∆CPT1A embryos. d, Genotyping for the presence of the Cpt1a floxed (2,200 bp) and excision band (300 bp) at E11.5. e, LYVE1+ dorsal dermal lymphatic vessels. White dashed lines: left and right lymphatic front. Bottom panels: high magnification insets of red boxed region. Note that LYVE1 also stains macrophages. Scale bars, 500 μm. f, Quantitation of the lymphatic vessel outgrowth towards the midline, as measured by the average remaining gap between left and right flank lymphatics (n = 7 embryos for Flt4; n = 8 embryos for Flt4∆CPT1A; 2 litters). g–i, Quantitation of the total vessel length (normalized per total area) (g), number of branch points (corrected for lymphatic length) (h) and average vessel width (i) (n = 7 embryos for Flt4; n = 8 embryos for Flt4∆CPT1A; 2 litters). j–x, Embryonic phenotype of vehicle- (ctrl) or etomoxir-treated (eto) embryos. j, Representative stereomicrographs at E14.5. Asterisks denote subdermal oedema. k, VEGFR3 immunostaining in the jugular region of E14.5 embryos. Solid white line, jugular vein (JV); dashed white line, JLS. Scale bars, 50 μm. l, JLS area in E14.5 embryos (n = 8 embryos for ctrl; n = 5 embryos for eto; 1 litter for each group). m–s, Analysis of E16.5 ctrl- or eto-treated embryos. m, LYVE1+ dermal lymphatic vessels. White dashed lines, indicate the left and right lymphatic front. Scale bars, 500 μm. n–q, Lymphatic vessel outgrowth (n), total lymphatic vessel length (normalized per total image area; o), number of lymphatic branch points (corrected for lymphatic length; p), and average lymphatic vessel width (q) (n = 6 embryos for ctrl; n = 6 embryos for eto; 2 litters). r, Quantitation of the number of filopodia per lymphatic sprout tip (n = 5 embryos for ctrl; n = 6 embryos for eto; 2 litters per group). s, The number of proliferating BrdU+ LECs in E15.5 ctrl and etomoxir (eto) embryos (n = 4 embryos for ctrl; n = 6 embryos for eto; 1 litter per group). t, Quantitation of the vascular area in primary head vein (left and right side) and the internal carotid artery (rostral extension of the dorsal aorta; left and right side) in E10.5 ctrl- and eto-treated embryos. u–x, Quantitation of the lymphatic vessel outgrowth (u), total lymphatic vessel length (v), the number of lymphatic branch points (w), and average lymphatic vessel width (x) in the dorsal dermal lymphatic network in E15.5 embryos treated from E8.5–E14.5 with vehicle (ctrlE8.5–E14.5) or etomoxir (etoE8.5–E14.5) (n = 6 embryos for ctrlE8.5–E14.5; n = 5 embryos for etoE8.5–E14.5). Mean ± s.e.m. Statistical test, ANOVA and Bonferroni post-hoc test were used in multiple group comparisons. t-test was used for comparison of two groups. *P < 0.05; NS, not statistically significant.