mbtps1uq28bh and sec23auq22bh embryos fail to form a lymphatic network in the trunk. (A-D) Confocal images of Tg(fli1a:nEGFP); Tg(-5.2lyve1b:dsRed) (green/magenta) sibling (A,C), mbtps1uq28bh (B) and sec23auq22bh (D) embryos at 5 dpf showing mbtps1uq28bh and sec23auq22bh lack TD, DLLV and ISLVs at 5 dpf. Absence indicated by asterisks. All embryos show normal development of DA, PCV and vISV at 5 dpf. (E,F) Quantification reveals fewer LECs within vessels in mbtps1uq28bh (E) and sec23auq22bh (F) when compared with siblings. Measured across five somites (E) and eight somites (F). (G,H) At 54 hpf, there are fewer PLs at the HM in mbtps1uq28bh (G) and sec23auq22bh (H) mutants when compared with siblings (measured across five somites). (I) At 3 dpf, there are no significant differences in the number of PLs at the HM of sec23auq22bh mutants when compared with siblings (measured across five somites). (J,K) At 5 dpf there are significantly more PLs at the HM in mbtps1uq28bh (J) and sec23auq22bh (K) mutants when compared with siblings (measured across five somites). (L,M) Time-lapse imaging of Tg(fli1a:nEGFP); Tg(-5.2lyve1b:dsRed) showing PLs migrate from the HM in siblings (representative of n=7 embryos analysed) when compared with sec23auq22bh mutants (representative of n=4 embryos analysed). Yellow arrow indicates migrating PL (L) and stalled PL (M). (N) Cross-section showing PLs migrate adjacent to notochord. (O) There were significantly fewer PLs migrating in sec23auq22bh mutants (n=4) when compared with siblings (n=7). ns, not significant. *P<0.05, ***P<0.001, ****P<0.0001 (two-tailed unpaired Student's t-test). DA, dorsal aorta; DLLV, dorsal longitudinal lymphatic vessel; ISLVs, intersegmental lymphatic vessels; N, notochord; PCV, posterior cardinal vein; PL, parachordal LEC; TD, thoracic duct; vISV, venous intersegmental vessel. Scale bars: 50 µm in A-D,L,M; 80 µm in N.
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