Ny A , Vandevelde W , Hohensinner P , Beerens M , Geudens I , Diez-Juan A , Brepoels K , Plaisance S , Krieg PA , Langenberg T , Vinckier S , Luttun A , Carmeliet P , Dewerchin M .

	Fig. 1. GFP expression in blood and lymphatic vessels of Tg(Flk1:eGFP) tadpoles. All panels depict lateral views of the tadpoles, head facing left. (A) Stage 40�45 Tg(Flk1:eGFP) tadpole showing GFP signal in the entire blood and lymphatic vasculature. (B) Higher magnification of fluorescent LH (encircled) and connecting lymphatic vessels in a stage 45 tadpole. The region demarcated by the left square in panel A is shown. (C) Higher magnification of GFP+ blood and lymphatic vessels in the trunk of a stage 45 tadpole. The region demarcated by the right square in panel A is shown. (D) Angiography by intracardial injection of high molecular TRITC-dextran exclusively labeled the blood vasculature (shown in orange). (E) Reversely, lymphangiography by injecting the TRITC-dextran dye in the fin adjacent to the DCLV (black asterisk denotes site of injection), showing specific uptake of the dye by the lymphatics (shown in orange) and draining towards the lymph heart. Inset shows higher magnification of the zone proximal to the injection site. CLD, cephalic lymph duct; DA, dorsal aorta; DCLV, dorsal caudal lymph vessel; DLAV, dorsal longitudinal anastomosing vessel; ISV, intersomitic vessel; LH, lymph heart; LLD, lateral lymph duct; PCV, posterior cardinal vein; VCLV, ventral caudal lymphatic vessel. Scale bars: 500 �m (A), 200 �m (B�E).
	Fig. 2. In vivo labeling of lymphendothelial cells allows parallel blood- and lymph vessel analysis. (A,B) Specific �LEC labeling� of lymph vessels (orange) by TRITC-dextran 24 hours after angiography of stage 46 tadpoles, allowing prolonged visualization of the vessels in the lymph heart (asterisk) area (A) and tail (B). (C�C″) Confocal imaging of cross sections of a LEC labeled tadpole confirmed exclusive labeling of lymphatics (DCLV, VCLV), while blood vessels (PCV, DA) remain green. C′�C″ are higher magnifications of the dorsal (C′) and ventral (C″) frames in panel C. (D) High magnification of vascular sprouting into the fin showed distinct morphology between smooth blood vessels (green) and the spiky lymphatics (red/orange). (E,F) Blood vessel (PCV, green) and lymph vessel (VCLV, yellow/organge) in close proximity. LECs of the VCLV exhibit small protrusions and filopodia (arrowheads). (G�G″) Newly forming blood vessel sprouts display a typical tip cell phenotype (arrowheads) with several filopodia. G′�G″ are higher magnifications of angiogenic tip cells. (H) Lymphatic tip cell with protruding filopodia (arrowhead). (I,I′) High magnification of lymphatic tip cells with filopodia (arrowheads) at the forefront of a lymph vessel in the fin (I) or of a new sprout forming from the DCLV (I′). (J) Lymphatics (red) occasionally sprouted at the same site as blood vessels (green), seemingly using them as a scaffold for further elongation into the fin. CLD, cephalic lymph duct; DA, dorsal aorta; DCLV, dorsal caudal lymph vessel; LLD, lateral lymph duct; NC, notochord; PCV, posterior cardinal vein; VCLV, ventral caudal lymph vessel. Scale bars: 200 �m (A,B), 100 �m (C), 50 �m (C′,C″,D,G), 20 �m (E,F,H,I′,J), 10 �m (G′,G″,I).
	Fig. 3. Tg(Flk1:eGFP) transgenic tadpoles as a tool to study developmental lymphangiogenesis. All panels depict lateral views of stage 45 tadpoles, head facing left. All insets show higher magnification of the dorsal side, with top and lower bracket denoting DCLV and DLAV, respectively. (A�C) Control embryo showing normal morphology (A) and correctly formed GFP+ (B) and LEC labeled lymphatics (C). (D�F) Morpholino knockdown (KD) of xProx1 resulted in edema around heart, gut and cloaca (arrowheads in panel D). GFP fluorescent microscopy showing that Prox1KD tadpoles possess few and disorganized LECs failing to assemble into the dorsal and ventral lymphatics (E). LEC labeling showing fewer LECs in xProx1KD tadpoles. Arrowheads denote sites where longitudinal lymph vessels are missing or malformed (green: DLAV, orange: DCLV) (F). (G�I) Morpholino knockdown (KD) of xVEGF-C resulted in edema in the heart and gut (arrowheads in panel G). Reporter VEGF-CKD tadpoles revealing a fragmented DCLV consisting of dispersed and scattered LECs on the dorsal side while the VCLV appears grossly normal as shown by fluorescent microscopy (H) and LEC labeling (I). Arrowheads denote sites where longitudinal lymph vessel is missing. (J�L) Chemical inhibition of VEGFR-3 by MAZ51 treatment (10 �M) resulted in edema in the heart and gut (arrowheads in panel J). Fluorescent microscopy (K) and LEC labeling (L) revealing a disorganized and fragmented DCLV consisting of few LECs, while the VCLV appeared normal. Arrowheads denote where longitudinal lymph vessels are missing. DA, dorsal aorta; DCLV, dorsal caudal lymph vessel; DLAV, dorsal longitudinal anastomosing vessel; PCV, posterior cardinal vein; VCLV, ventral caudal lymph vessel. Scale bars: 1 mm (A,D,G,J), 250 �m (B,E,H,K).
	Fig. 4. Modulation of lymphatic sprouting. All photos depict lateral views of LEC labeled stage 49 Tg(Flk1:eGFP) tadpoles (blood vessels green, lymph vessels orange), head facing left. (A�C) Representative micrographs showing inhibition of lymphatic sprouting into the fin (arrowheads) upon MAZ51 treatment (5 �M) (B), as compared to DMSO treated controls (A). Quantification of lymphatic sprout numbers in panel C (*P<0.001 compared to DMSO control). (D�J) Representative micrographs of effect of GFP (control) (D), VEGF-A (E) and VEGF-C (F) overexpression on blood and lymph vessel sprouting after injection of transfected cells in the fin. Asterisk denotes the location of the cell cluster. Quantification of the number of blood vessel sprouts (G), lymph vessel sprouts (H), total blood vessel length (I) and total lymph vessel length (J) (*P<0.001 compared to GFP control, #P<0.001 compared to VEGF-A overexpression). (K�M) Chemical inhibition of VEGF-C induced lymphatic sprouting by MAZ51. Representative micrographs of tadpoles injected with VEGF-C overexpressing cells and treated with DMSO (K) or MAZ51 (L). Corresponding control with GFP overexpressing cells (quantified in panel M) is not shown. Asterisk denotes the location of the cell cluster. Quantification of the number of lymphatic sprouts after MAZ51 treatment in panel M (*P<0.001 compared to GFP control, #P<0.001 compared to VEGF-C overexpression). Scale bars: 500 �m (A,B), 250 �m (D�F,K,L). Quantitative data are mean � s.e.m.
	Fig. S3. Development of the blood- and lymphatic vascular network in the fin. All panels depict lateral views of the tadpoles, head facing left. Each fluorescence picture (A�D) is accompanied by a schematic redrawing of the blood vascular sprouts (green) and lymphatic sprouts (red) (A9�D9). A single tadpole was followed during the indicated stages. (A,A9) First blood vessel sprouting into the ventral fin is seen from stage 46 (6 days post fertilization (dpf)) onwards. (B,B9) Blood vessel sprouting is followed by lymphatic sprouting into the ventral fin, commencing at stage 47. Denoted by arrowheads, already formed blood vessels turning back towards the PCV to form a closed loop (8 dpf). (C,C9) Start of vessel sprouting into the dorsal fin (12 dpf). (D,D9) No or very few new blood and lymphatic sprouts are formed from the longitudinal vessels from this stage onwards (15 dpf and older). Instead, expansion of the dorsal and ventral vascular network depends on the growth of the already established primary sprouts and the formation of new secondary sprouts. Scale bars: 0.5 mm.
	Fig. S4. Cell cluster formation after cell injection into the fin. Example of an injection of cells into the fin showing the formation of a cell cluster at the injection site. Injected cells remain clearly visible under the microscope for up to 1 week. Only tadpoles with comparable cell cluster size were used in the experimental analysis. Scale bar: 200 mm.

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