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Fig. 4 Abnormal migratory dynamics of PLs underpins an absence of lymphatic vessels in col2a1auq36bh mutant embryos. (A) Quantification of migration distance along the anterior-posterior (a-p) axis for mutant and sibling PLs (n=6 embryos). (B) Quantification of the anterior-posterior (a-p) axis PL migration velocity over 20 min in sibling and mutant embryos (n=6 embryos). (C) Quantification of the sphericity of PL nuclei in µm2 at 3 dpf and 4 dpf in col2a1auq36bh mutants compared with siblings. (D) Quantification of the dorso-ventral axis migration for sibling and mutant PLs (n=6 embryos). (E,F) Time-lapse imaging from 60 to 80 hpf of Tg(fli1a:nEGFP); Tg(-5.2lyve1b:dsRed) showing a PL migrating from the HM in siblings (n=6) compared with col2a1auq36bh mutants (n=6). The nuclei of a migrating (E) and stalled (F) PL are rendered blue (and indicated by blue arrow). (G,H) Quantification of dorso-ventral migration showing individual cell tracks (G) and mean±s.d. (H) for sibling and col2a1auq36bh PLs. (I-L) Confocal images of Tg(-5.2lyve1b:dsRed) (magenta) in uninjected siblings (I, n=20) compared with sec23auq22bh mutants (white asterisks, J, n=29) and embryos injected with pDest:Col2a1:sec23a-p2a-EGFP (green) (K,L). sec23auq22bh mutants displayed a partial rescue of TD development (white arrows) (L, n=21). Injected siblings (K, n=14) and sec23auq22bh mutants showed formation of ectopic vessels (yellow arrows). (M) Quantification of ectopic fragment formation in injected embryos compared with uninjected controls for both sibling and sec23auq22bh mutants. (N) Quantification of TD formation in sec23auq22bh mutants injected with col2a1a-Sec23a-T2A-eGFP. In siblings, the formation of ectopic fragments leads to reduced TD formation. (O,P) Schematic showing a model for Col2a1 secretion from NSCs into the ECM around the migrating PLs (O). Defects in Col2a1 secretion from NSCs leads to failed migration of PLs (P). ns, not significant. *P<0.05, ***P<0.001, ****P<0.0001 (two-tailed unpaired Student's t-test). HM, horizontal myoseptum; M, myotome; N, notochord; NT, neural tube; PL, parachordal LEC.