Extended Data Figure 3 : Early and later-stage lymphatic-specific loss of CPT1A results in defects in lymphatic development.

From: The role of fatty acid β-oxidation in lymphangiogenesis

Extended Data Figure 3

a, Representative stereomicrographs of an E14.5 Prox1 (left) and a Prox1∆CPT1A embryo with deletion of Cpt1a in PROX1-positive endothelial cells (right). Asterisks denote subdermal oedema. Arrows highlight subdermal bleeding spots. b, Zoomed in micrograph of the Prox1∆CPT1A embryo shown in a (right). Arrows denote bleeding spots. c, Representative micrograph of TER119 immunostaining (green) of erythroid precursors (arrows) present within the JLS of a Prox1∆CPT1A embryo, as highlighted by LYVE1 staining (red). Dotted lines denote endothelium in lymphatic structures. Scale bar, 100 μm. d, Zoomed in micrograph of lymphatic capillaries of an E16.5 Prox1∆CPT1A embryo, visualized by Evans blue dye injection (blue) in the forepaws. Arrows indicate blood-filled blunt-ended dermal lymphatic structures. eo, Experiments involving the anterior dorsal dermal lymphatic vessels of E15.5 wild-type (Prox1) and Prox1∆CPT1A embryos. e, Immunostaining for LYVE1+ lymphatic vessels. White dashed lines: left and right lymphatic front. Lower panels: high magnification images of red, boxed regions. Scale bars, 500 μm. f, Quantitation of the lymphatic vessel outgrowth towards the midline in the anterior dorsal back skin, as measured by the average remaining gap between left and right flank lymphatics (n = 6 embryos for wild type; n = 9 embryos for Prox1∆CPT1A; 3 litters). gi, Quantification of the total vessel length relative to the total field area (g), number of branch points (corrected for lymphatic length) (h) and average vessel width (i) (n = 6 embryos for wild type; n = 9 embryos for Prox1∆CPT1A; 3 litters). j, LYVE1+ dermal lymphatic vessels in an E15.5 Prox1∆CPT1A embryo or an E16.5 Prox1FK∆CPT1A embryo highlighting the presence of lymphatic islands (white arrows). Scale bars, 500 μm. km, Experiments involving the anterior dorsal dermal lymphatic vessels of E15.5 wild-type (Prox1) and Prox1∆CPT1A embryos treated with tamoxifen from E10.5–E12.5 to inactivate Cpt1a in LECs after initial lymphatic structure formation has occurred. k, Immunostaining for LYVE1+ lymphatic vessels. White dashed lines, left and right lymphatic front. Lower panels, high magnification images of red boxed regions. Scale bars, 500 μm. l, Quantitation of the lymphatic vessel outgrowth towards the midline in the anterior dorsal back skin, as measured by the average remaining gap between left and right flank lymphatics (n = 5 embryos for wild type; n = 6 embryos for Prox1∆CPT1A; 2 litters). m, Quantification of the total vessel length relative to the total field area (n = 5 embryos for wild type; n = 6 embryos for Prox1∆CPT1A; 2 litters). np, Experiments involving the anterior dorsal dermal lymphatic network in E15.5 wild-type (Prox1) and Prox1∆CPT1A embryos. n, High magnification images of VEGFR3+ (green) lymphatic sprouts. The white arrows indicate filopodia. Note the weaker VEGFR3 immunoreactive signal in the Prox1∆CPT1A embryo (right panel). Inset panels represent LYVE1 staining, confirming the identification of filopodia by another marker (note that LYVE1 also stains macrophages, which are sometimes in the proximity of lymphatic sprouts, but clearly distinguishable based on their morphology). Scale bars, 10 μm. o, Number of filopodia per lymphatic sprout tip (n = 6 embryos for wild type; n = 9 embryos for Prox1∆CPT1A; 3 litters). p, VEGFR3 (white) immunoreactivity in the growing dorsal dermal lymphatic vessels. Note the weaker VEGFR3 immunoreactive signal in the Prox1∆CPT1A embryo (right panel). Scale bars, 100 μm. Mean ± s.e.m. Statistical test: t-test was used for comparison of two groups. *P < 0.05.