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Supplementary Material

DEV077701 Supplementary Material

Files in this Data Supplement:

  • Supplemental Figure S1 -

    Fig. S1. The lyve1 promoter marks regions of endogenous lyve1 expression and labelled trunk lymphatics develop in a pattern known to occur in the emergence of the thoracic duct. (A-L) lyve1 probe (A-D), gfp probe (E-H) and lyve1 (BM Purple) gfp (Fast Red) probes (I-L) applied to lyve1:EGFP embryos at 1 dpf (A,B,E,F,I,J) and 2 dpf (C,D,G,H,K,L) (A,C,E,G,I,K, lateral view; B,D,F,H,J,L dorsal view of anterior region). Black arrows indicate areas where gfp expression does not match endogenous lyve1 expression. (M-M′′) Lateral image of a lyve1:DsRed2;SAGFF27C;UAS:YFP transgenic at 5 dpf showing SAGFF27C;UAS:YFP expression (M), lyve1:DsRed2 (M′) and overlapping lyve1:DsRed2;SAGFF27C;UAS:YFP expression in the trunk lymphatic vessels (M′′). (N-N′′) Lateral image of a lyve1:EGFP;lyve1:DsRed2 transgenic at 5 dpf showing lyve1:EGFP expression (N), lyve1:DsRed2 (N′) and overlapping lyve1:EGFP;lyve1:DsRed2 expression in the trunk lymphatic vessels and PCV (N′′). (O-Q) Lateral images of the developing trunk lymphatics in the lyve1:EGFP transgenic at 1.3 dpf (O), 2 dpf (P) and 3 dpf (Q). (R,R′) Lateral image of the developed trunk lymphatics in lyve1:EGFP transgenic at 6 dpf (R). Microangiography confirms that these vessels contain no blood-flow (R′). DA, dorsal aorta; DLLV, dorsal longitudinal lymphatic vessel; ISLV, intersegmental lymphatic vessel; ISV, intersegmental vessel; LSS, lymphatic secondary sprout; PCV, posterior cardinal vein; PL, parachordal lymphangioblasts; TD, thoracic duct; vISV, venous intersegmental vessel. Scale bar: 100 µm.

  • Supplemental Figure S2 -

    Fig. S2. Atlas of lymphatic development in zebrafish from 2 dpf to 15 dpf. (A-H′) Montage of maximum projections of iz series stacks of the entire lyve1:DsRed2;kdrl:EGFP and lyve1:EGFP;kdrl:RFP transgenics (lateral views) at 2-7 dpf (A-F), 12 dpf (G) and 15 dpf (H), and their corresponding ventral images of the head and the anterior intestine (A′-H′). DLLV, dorsal longitudinal lymphatic vessel; ICLV, intercostal lymphatic vessel; ISLV, intersegmental lymphatic vessel; FLS, facial lymphatic sprout; LAA1, first lymphatic branchial arch; LAA2, second lymphatic branchial arch; LAA3, third lymphatic branchial arch; LAA4, fourth lymphatic branchial arch; LFL, lateral facial lymphatic; LL-IL, lower-left intestinal lymphatic; LR-IL, lower-right intestinal lymphatic; L-SIL, left supraintestinal vessel; PHS, primary head sinus; PLV, parachordal lymphatic vessel; MFL, medial facial lymphatic; OLV, otolithic lymphatic vessel; TD, thoracic duct; UL-IL, upper-left intestinal lymphatic; UR-IL, upper-right intestinal lymphatic. Scale bars: 300 µm.

  • Supplemental Figure S3 -

    Fig. S3. The lymphatic branchial arches develop along the arterial branchial arches. (A,A′) Ventrolateral images of vessels of the pharyngeal area in the lyve1:DsRed2;kdrl:EGFP transgenic at 7 dpf (A), with schematic diagram of arteries (red), veins (blue) and lymphatics (green) (A'). (B,B′) Ventral image of head vessels in the lyve1:DsRed2;kdrl:EGFP transgenic at 7 dpf (B), with schematic diagram (B'). A and B are montage images of two z series stacks. AA3, first branchial arch; AA4, second branchial arch; AA5, third branchial arch; AA6, fourth branchial arch; LAA1, first lymphatic branchial arch; LAA2, second lymphatic branchial arch; LAA3, third lymphatic branchial arch; LAA4, fourth lymphatic branchial arch; LFL, lateral facial lymphatic; MFL, medial facial lymphatic. Scale bars: 100 µm.

  • Supplemental Figure S4 -

    Fig. S4. Functional characterisation of the facial lymphatics. (A-A′′) Lateral images of the lyve1:DsRed2 transgenic at 8 dpf showing the FL (A), its dye uptake a few minutes after subcutaneous injection of high molecular weight fluorescein dextran (n=10) (A′), and the merged image of A and A' (A′′). (B-B′′) Lateral images of lyve1 (B), gata1 (B′) and merged (B′′) expression of the lyve1:EGFP:gata1:DsRed transgenic at 5 dpf showing no blood flow in the FL. (C) The lack of blood flow is more clear in ventral confocal image of the lyve1:EGFP:gata1:DsRed transgenic at 5 dpf. (B-C) Montage images of two z series stacks. FL, facial lymphatics. Scale bars: 100 µm.

  • Supplemental Figure S5 -

    Fig. S5. The ventral aorta lymphangioblast is derived from a non-arterial vessel near the ventral aorta. (A,A′) 3D reconstruction of the region where VA-L originates in the lyve1:EGFP;kdrl:RFP transgenic at 1.5 dpf. The VA-L co-expresses kdrl indicating its vascular origins. (B,B′) Ventrolateral images of the kdrl:RFP;flt1enh:YFP transgenic at 1.5dpf. The VA-L does not have flt1enh:YFP expression (B′) indicating its non-arterial origin (n=13). VA, ventral aorta; VA-L, ventral aorta lymphangioblast. Scale bars: 50 µm.

  • Supplemental Figure S6 -

    Fig. S6. Ventral aorta lymphangioblast migration requires flt4 and ccbe1. (A-D) flt4 probe (A,B) and vegfc probe (C,D) applied to embryos at 1 dpf (A,C) and 2 dpf (B,D) showing flt4 and vegfc expression in the head at 2 dpf (D). (E) Quantification of the length of the VA-L in control (n=22), flt4 (n=32) and ccbe1 (n=26) morpholino-injected embryos at 2 dpf. Error bar represents 95% confidence interval. ***P<0.001. VA-L, ventral aorta lymphangioblast.

  • Movie 1 -

    Movie 1. The connection between the thoracic duct and the supraintestinal lymphatics. Movie of a confocal z stack, showing the connection (indicated by white asterisks) between the thoracic duct, and the right and left supraintestinal lymphatic vessels (R-SIL and L-SIL) at 5 dpf. Movie consists of 45 z slices, each 5 µm apart, played at five frames per second. L-SIL, left supraintestinal lymphatic vessel; PCV, posterior cardinal vein; R-SIL, right-supraintestinal lymphatic vessel. Scale bar: 100 µm.

  • Movie 2 -

    Movie 2. Time-lapse movie of the development of the lateral facial lymphatic from 1.63-2 dpf. Movie of the lyve1:EGFP;kdrl:nlsmCherry transgenic from 1.63 dpf to 2 dpf (39 hpf to 48 hpf, 9 hours total). The lyve1 channel (right) shows the migration of the FLS from the common cardinal vein and the recruitment of PHS-Ls to the vascular tip of the FLS. The nuclear kdrl channel (left) shows lymphangioblast cell duplication (indicated by yellow and red asterisks) within the FLS, and the migration of lymphangioblast cells (indicated by asterisks). Time-lapse image stacks were collected every 10 minutes. Movie was made at seven frames per second. FLS, facial lymphatic sprout; PHS-L, primary head sinus lymphangioblast. Scale bar: 50 µm.

  • Movie 3 -

    Movie 3. Time-lapse movie of the development of the lateral facial lymphatic from 2-2.5 dpf. Movie of the lyve1:EGFP;kdrl:nlsmCherry transgenic from 2 dpf to 2.5 dpf (48 hpf to 60 hpf, 12 hours total). The same embryo was used as in Movie 1. The lyve1 channel (right) shows the migration of the FLS, and the recruitment of the final PHS-L and the VA-L to the vascular tip of the FLS to form the lateral facial lymphatic. The nuclear kdrl channel (left) shows lymphangioblast cell duplication (indicated by green and light blue asterisks) within the FLS, and the migration of lymphangioblast cells (indicated by asterisks) from the FLS to another PHS-L and the VA-L. Time-lapse image stacks were collected every 10 minutes. Movie was made at seven frames per second. FLS, facial lymphatic sprout; PHS-L, primary head sinus lymphangioblast; VA-L, ventral aorta lymphangioblast. Scale bar: 50 µm.

  • Movie 4 -

    Move 4. A flt4 morphant embryo fails to form the facial lymphatic sprout. Movie showing flt4 (left) and control (right) morpholino-injected lyve1:EGFP embryos from 1.63 dpf to 2 dpf (39 hpf to 48 hpf, 9 hours total). The FLS (indicated by yellow asterisk) fails to form in the flt4 morphant embryo. Despite the lack of FLS, the pectoral fin vessel and the posterior cerebral vein (indicated by red and white asterisks respectively) still forms in the flt4 morphant, showing that blood vessel development is proceeding normally in this embryo. Time-lapse image stacks were collected every 10 minutes. Movie was made at seven frames per second. Scale bar: 50 µm.