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Fig. 2 iYSN converge by cortical flow within the YSL. (A-C′) Microtubules labeled with Tau-GFP (A), microtubule plus-ends labeled with EB3-GFP (B) and actin labeled with Lifeact-GFP (C,C′) in the paraxial region of the iYSL at 90% epiboly (9 hpf; A,C) and bud stage (10 hpf; B). Asterisks mark iYSN. (C′) Dotty actin staining in more superficial layers of the YSL and filamentous actin in deeper layers (C). (D-F) Vector fields showing optical flow direction (yellow arrows) of Tau-GFP (D), EB3-GFP (E) and Lifeact-GFP (F). Red arrows indicate the movement direction of the iYSN. (G) Two-photon image of two-somite stage wild-type embryo with fluorescently labeled YSN and 0.5 μm diameter fluorescent microspheres injected into the YSL. Image is a z-projection. Several nuclear (arrowheads) and microsphere (bead) trajectories (arrows) obtained using Motion Tracking Software are shown. Circles indicate the endpoint of each track. The white line indicates the dorsal midline of the embryo. (H) Comparison of the speed of YSN and bead movements in control embryos (n=6 embryos) and embryos injected with beads (n=2 embryos). No significant differences were found in average and net dorsal speed of YSN and beads (P>0.05). Notably, injected beads appear to have slightly less persistent movements compared with adjacent iYSN (see Movie 4 in the supplementary material). This reduction in bead persistence is probably due to the smaller diameter of the beads (0.5 μm) compared with iYSN (6-20 μm), resulting in greater diffusive relative to advective motion for beads in comparison with iYSN. Dorsal is towards the right (A-F) and animal is towards the top (A-G). Scale bars: 10 μm in A-C; 50 μm in G.