The intestinal lymphatics span the entire intestine and its development is closely associated with the intestinal blood vasculature. All the images used in this figure are generated from the lyve1:DsRed2;kdrl:EGFP transgenic. (A,A′) Lateral image of the right-hand side of the embryo showing the initial lyve1-positive vessel (indicated by asterisks) forming along the AMA, SIA and the PA at 4 dpf (A), with schematic diagram of arteries (red), veins (blue) and lymphatics (green) (A′). (B,B′) Dorsolateral image of the right side of the embryo showing the UR-IL and the LR-IL developing along the SIA and the R-SIV, respectively (B), with schematic diagram (B′). (C,C′) Lateral image of the left side of the embryo showing the UL-IL and the LL-IL that develops along the L-SIV (C), with schematic diagram (C′). Asterisks indicate the connection between the L-IL and the R-IL. The posterior regions of the SIA and the UR-IL are also visible. (D) Dorsal view of the intestinal vessels at 5 dpf. Dotted green lines represent proposed vessel connections between the L-IL and the R-IL. (E,E′) Lateral image showing the connections (indicated by asterisks) between the SILs and the TD at 5 dpf. Blue and black asterisks represent connections between the R-SIL and L-SIL with the TD, respectively. (F,F′) Ventral image showing a pair of LR-IL that forms in close proximity along the R-SIV at 6 dpf (F), with schematic diagram (F′). (G-H′) Lateral images of the right (G), and the left (H) side of the embryo showing the intestinal vessels at 7 dpf, with schematic diagrams (G′,H′). The L-IL branch is labelled with an asterisk in H and H′. (I,I′) Lateral image of the left side of the embryo showing the L-IL branches (indicated by asterisks) and branches extending from the R-IL to the left side of the intestine (indicated by pink arrows) at 15 dpf (I), with schematic diagram (I′). (J) Quantification of the number of L-IL branches at 7 dpf (average 1.3, n=22) and 15 dpf (average 4.4, n=10). Error bar represents 95% confidence interval. ^***P<0.001. (K,K′) Lateral image of the right side of the embryo showing the intestinal vessels at 15 dpf (K), with schematic diagram (K′). Lymphatic vessels whose positions are variable between individual embryos are in pink. Connection between the L-IL and the R-IL is indicated with an asterisk. (L,L′) Lateral image showing the SIL branches (indicated by asterisks) at 15 dpf (L), with schematic diagram (L′). Only the lyve1:DsRed2 expression was shown for clarity. B,E,G,H,I,K,L are montage images. B,E,G,H,I used two z series stacks. K used six z series stacks. L used four z series stacks. AMA, anterior mesenteric artery; CCV, common cardinal vein; DA, dorsal aorta; DLLV, dorsal longitudinal lymphatic vessel; ISLV, intersegmental lymphatic vessel; L-IL, left intestinal lymphatics; LL-IL, lower-left intestinal lymphatic; LR-IL, lower-right intestinal lymphatic; L-SIL, left supraintestinal lymphatic vessel; L-SIV, left subintestinal vein; PA, pancreatic anlage; PCV, posterior cardinal vein; R-IL, right intestinal lymphatics; R-SIV, right subintestinal vein; SIA, supraintestinal artery; TD, thoracic duct; UL-IL, upper-left intestinal lymphatic; UR-IL, upper-right intestinal lymphatic. Scale bars: 100 μm in A-H; 200 μm in I-L.

The lateral lymphatics sprout from the intersegmental lymphatic vessel and form along the parachordal vessel and the intercostal vessel. All the images used in this figure are generated from the lyve1:DsRed2;kdrl:EGFP transgenic. (A) Lateral image of the trunk vessels at 5 dpf. (B) 3D reconstruction of the white box in A showing the initial sprout of the PLV originating from the ISLV. (C) Lateral image of the trunk vessels at 7 dpf, showing fusion of the PLV fragments. (D) Dorsal image of the trunk vessels at 15 dpf. (E) 3D reconstruction of the trunk vessels at 15 dpf, showing the dorsal migration of the ICLVs along the ICV. (F) Lateral image of the lateral lymphatics at 15 dpf. F is a montage image of eight z series stacks. DA, dorsal aorta; ICV, intercostal vessel; ICLV, intercostal lymphatic vessel; ISLV, intersegmental lymphatic vessel; ISV, intersegmental vessel; PAV, parachordal vessel; PCV, posterior cardinal vein; PLV, parachordal lymphatic vessel; TD, thoracic duct. Scale bars: 100 μm in A,C,D; 200 μm in F.

Distinct lymphangioblast populations contribute to the development of the LFL. (A-D″) Lateral images of lyve1:EGFP expression (A-D) and both lyve1:EGFP and kdrl:RFP expression (A′-D′) of the lyve1:EGFP;kdrl:RFP transgenic at 1.5 dpf (A), 1.75 dpf (B), 2 dpf (C) and 2.5 dpf (D), with schematic diagrams of arteries (red), veins (blue) and lymphatic (green) at each stage (A″-D″). Lymphangioblast populations are indicated by asterisks. The PHS-L is in pink; the VA-L is in grey. AA1, mandibular arch; CCV, common cardinal vein; FLS, facial lymphatic sprout; LFL, lateral facial lymphatic; PHS, primary head sinus; PHS-L, primary head sinus lymphangioblast; VA, ventral aorta; VA-L, ventral aorta lymphangioblast. Scale bars: 100 μm.

The LFL develops from a vascular sprout with migration, duplication, and recruitment of lymphangioblast populations to the vascular tip. (A,A′) Still images from supplementary material Movie 2 showing the developing LFL in the lyve1:EGFP;kdrl:nlsmCherry transgenic from 1.63 to 2 dpf (39 to 48 hpf). (A) lyve1 expression shows the vascular tip of the FLS, which resembles a dynamic vascular sprout with filopodia (red arrow) and lamellipodia (yellow arrow), and the migration and the recruitment of PHS lymphangioblasts (indicated by a white asterisk) to the vascular tip of the FLS. (A′) Nuclear kdrl expression shows duplication of a lymphangioblast cell (yellow and red) within the FLS (green outline), the migration of lymphangioblast cells (yellow, red, blue and beige) and the recruitment of PHS-Ls (purple outline). (B,B′) Still images from supplementary material Movie 3 showing the developing LFL in the same lyve1:EGFP;kdrl:nlsmCherry transgenic embryo as in A from 2 to 2.5 dpf (48 to 60 hpf). (B) lyve1 expression shows the migration and recruitment of another PHS-L (white asterisk) and the VA lymphangioblast (yellow asterisk) to the growing tip of the FLS to form the LFL. (B′) Nuclear kdrl expression shows duplication of lymphangioblast cells (green and light blue) within the FLS (green outline), and the migration of lymphangioblast cells from the PHS-L that have been recruited to the FLS (orange, green and light blue) to another PHS lymphangioblast (pink outline) and the VA lymphangioblast (red outline). CCV, common cardinal vein; FLS, facial lymphatic sprout; LFL, lateral facial lymphatic; PHS, primary head sinus; PHS-L, primary head sinus lymphangioblast. Scale bars: 50 μm.

The development of the FL, IL and LL requires flt4 and ccbe1. (A-C) Lateral images of the lyve1:EGFP transgenic, showing altered FL and trunk lymphatics development in flt4 (B) and ccbe1 (C) morpholino-injected embryos compared with control morpholino-injected embryos (A) at 2-5 dpf. (D) Quantification of the length of the developing LFL in control, flt4 and ccbe1 morpholino-injected embryos at 2, 3 and 4 days post fertilisation. Error bars represent 95% confidence intervals. ^***P<0.001. (E-G′) Lateral images of the lyve1:DsRed2;kdrl:EGFP transgenic showing lack of IL and LL development in flt4 (F) and ccbe1 (G) morpholino-injected embryos compared with the normal IL and LL development in the control morpholino-injected embryos (E). E′-G′ are higher magnification images of the boxes in E-G. E-G are montage images of two z series stacks. DLLV, dorsal longitudinal lymphatic vessel; FLS, facial lymphatic sprout; ISLV, intersegmental lymphatic vessel; LFL, lateral facial lymphatic; PCeV, posterior cerebral vein; PCV, posterior cardinal vein; PLV, parachordal lymphatic vessel; R-IL, right intestinal lymphatics; R-SIV, right subintestinal vein; SIA, supraintestinal artery; TD, thoracic duct. VA-L, ventral aorta lymphangioblasts. Scale bars: 100 μm.