2°NC cells show distal-oriented movement after dissolving the cluster.

(A, B) Movement angle histogram for NC cells destined for proximal (A, 1°NC cells; n = 44) and distal (B, 2°NC cells; n = 37) sides of the eye. Movement trajectories of an individual NC cell were projected on a horizontal plane and the angle relative to the anteroposterior axis was measured every 10 time points (every 3 min 24 s). Each bin is 10°. Black arrows show median direction (56.5° and 83.6° for proximal and distal selection, respectively). Wallraff test of angular distances between two groups showed significant difference, p-value < 2.2x10-16. (C, D) Centred tracks starting from 22 hpf for proximal (C, 1°NC; n = 44) and distal (D, 2°NC; n = 37) destined NC cells. The starting position is set to 0 (bottom centre). (C) NC cells on the proximal side of the eye show moderate distal-oriented movement after 22 hpf. (D) In contrast NC cells destined for the distal side of the eye show higher displacement toward the distal end. (E-K) Cell morphology of 1°NC and 2°NC cells. (E-F) A 18-hpf embryo from Tg(sox10:mem-tdEosFP), highlighting NC cells in green. (E) 3D-overview of the optic cup region (stippled circle). (F) Projected focal planes for the proximal side of the eye (stippled region). Note the cuboidal or hexagonal cell shape of 1°NC cells retaining large areas of cell-cell contact with neighbouring cells. (G-K) Overview of a 26-hpf Tg(sox10:mem-tdEosFP) embryo (G, maximum projection) and magnified single cell morphology of NC cells in a single focal plane for proximal (H-I) and distal (J-K) ocular NC cells. Squares in G show individual locations of magnified cells in H-K. 1°NC cells appear flat and rectangular (arrows), while 2°NC cells show mesenchymal morphology with filopodia-like cellular protrusions (arrowheads). Orientation: dorsal (D), ventral (V), rostral (R) and caudal (C); Ln: lens; Scale bars: 20 μm. (PDF)