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Fig. 2 Parallel microtubule arrays are directed towards embryonic dorsal. (A,B) Immunostaining to detect α-Tubulin shows that parallel arrays form asymmetrically at one side of the vegetal cortex and cover a sector of ∼45°. Yellow dashed lines in A mark the extent of spread; yellow arrowhead in B marks one end of parallel arrays. (C) Parallel arrays are spread over ∼250 μm, similar to the spread of wnt8a transcripts at early cleavage stages. (D) Colocalization of parallel arrays (Dclk2-GFP in green) and fluorescent injected Alexa568 UTP-labeled wnt8a RNA (red punctae) at ∼20 mpf is shown. Inset shows higher magnification of area marked with white dotted square. (E-M) Injected fluorescent wnt8a RNA localizes asymmetrically to the vegetal lateral cortex (yellow arrowheads in I) and to the blastoderm (red asterisk in I), similar to endogenous wnt8a transcripts. (F-M) Orientation of parallel arrays at 20-30 mpf (F), compared with position of the dorsal organizer (shield, red arrow in G,H) at gastrulation (G), and the difference between orientation of parallel arrays and the position of the shield was used to calculate the deviation angle (δ) (H). More than 90% of embryos formed the shield within 30° of the parallel arrays (K) (n=23 embryos). EB1-GFP comets were tracked at 4-second intervals (E), starting at 20 mpf (L) or 35 mpf (M). Direction of movement of each comet was calculated and is presented as a purple arrow (E,J). 50 comets were tracked at 20 mpf and 30 comets at 35 mpf, n=6 embryos. The position of the shield was identified during gastrulation (black arrow in J), and the angle of deviation (δ) was calculated. Graph in L shows that most comets moved towards the future dorsal organizer at 20 mpf, whereas at 35 mpf (graph in M), comets at the same location moved randomly. A,D-H,J vegetal views; B,C,I, lateral views. Scale bars: in A-C,G,I, 100 μm; in D, 50 μm; in E, 1 μm; in F, 10 μm.