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Fig. 3 col2a1auq36bh mutants exhibit increased PL numbers in the HM over time and reduced numbers of LECs in mature vessels. (A,B) Confocal images of Tg(fli1a:nEGFP); Tg(-5.2lyve1b:dsRed) sibling (A, n=16) and col2a1a1uq36bh (B, n=9) at 54 hpf showing PLs migrate to the HM. (C-F) Confocal images of Tg(fli1a:nEGFP); Tg(-5.2lyve1b:DsRed) sibling (C, n=4; E, n=7) and col2a1auq36bh (D, n=5; F, n=7;) embryos at 3 dpf (C,D) and 4 dpf (E,F) show that PLs fail to migrate from the HM in col2a1auq36bh mutants compared with the siblings. (G,H) Confocal images of sibling (G, n=32) and col2a1auq36bh (H, n=23) embryos at 5 dpf showing stalled PLs at the HM in col2a1auq36bh mutants. Arrowheads indicate vessel fragments. (I,J) IF assay of Col2a1 and eGFP in Tg(fli1a:nEGFP); Tg(-5.2lyve1b:dsRed) siblings (I, thermal map I′, n=24) and col2a1auq36bh (J, thermal map J′, n=8) mutants confirm loss of Col2a1a in col2a1auq36bh. (K,L) No difference in PL number was observed in col2a1auq36bh and siblings at 54 hpf (K); however, at 5 dpf there was a significant increase in stalled PLs at the HM in col2a1auq36bh mutants when compared with siblings (L). (M) Quantification of nuclei in the TD and ISLV revealed a significant reduction in col2a1auq36bh (n=23) when compared with siblings (n=32). Line marked N indicates notochord. ns, not significant. *P<0.05, ***P<0.001 (two-tailed unpaired Student's t-test). TD, thoracic duct; HM, horizontal myoseptum; ISLVs, intersegmental lymphatic vessels; N, notochord; PL, parachordal LEC. Scale bars: 50 µm in A-H; 80 µm in I,J.