atoh8 (blue) and myoD expression (red) in the gastrula and early segmentation stages of development (6–12 hpf). a Shield stage (6 hpf). b 90% epiboly (9 hpf). b’, b’’ magnifications of image (b) showing dorsal and ventral atoh8 expression, respectively. c 9-hpf embryo double-stained for atoh8 and myoD. datoh8 expression at 9.5 hpf. e, e’ 10-hpf embryos stained for atoh8 alone and atoh8 and myoD, respectively; white arrowhead in e' indicates tail bud tissue free of atoh8 expression. f, f’ 10-hpf embryos stained for atoh8 alone and atoh8 and myoD, respectively. g results of RT-qPCR for atoh8 at developmental stages 2.5 to 11 hpf showing its expression relative to actb2. h, h’ Flat mount and schematic drawing of 10-hpf embryo showing atoh8 and myoD expression in blue and red, respectively. iatoh8 staining at 9.5 hpf. j, j’ 11.5-hpf embryos stained for atoh8 alone and atoh8 and myoD, respectively. k, k’’ 12-hpf embryos stained for atoh8. k’ 12-hpf embryo double-stained for atoh8 and myoD. l, l’ Flat mount and schematic drawing of 12-hpf embryo showing atoh8 and myoD expression in blue and red, respectively. Whole embryos: lateral view, anterior to the top, dorsal to the right. d’ ventral view (v); f, h, k, l dorsal view. Blue and red arrowheads: sites of atoh8 and myoD expression, respectively. Scale bars 200 µm

atoh8, myoD and crestin expression in early segmentation stages of development (13–14 hpf). a–eatoh8 ISH; f–hatoh8/myoD double ISH. i–katoh8/crestin double ISH. a Flat mount of 13.5-hpf embryo. b 13-hpf embryo showing first appearance of atoh8 in the developing hindbrain (dark arrowhead). c 13.5-hpf embryo. d Embryo at 14 hpf; pink arrowhead indicates the appearance of the second domain at the midbrain/hindbrain boundary. e1–e3 Sections through the 13-hpf embryo at the levels indicated in a. f, g Embryos at 13 hpf and 14 hpf, respectively, double-stained for atoh8 and myoD. h Flat mount of embryo at 14 hpf double-stained for atoh8 and myoD. i, j Flat mount and embryo showing double labelling for crestin and atoh8 at 13 hpf. k Embryo at 14 hpf double-stained for crestin and atoh8. a, h’ Dorsal view, anterior to the top. b–d, f, g, j, k lateral view, anterior to the top, dorsal to the right. h, i dorsal view, anterior to the left. Scale bars: whole embryos 200 µm, flat mounts a, h, i 200 µm; h’ 50 µm; transverse sections: 100 µm

atoh8, myoD and crestin expression profile in the 16–16.5-hpf embryo. a, b, f Whole embryos at 16.5 hpf, showing atoh8 and atoh8/myoD and atoh8/crestin signal distribution, respectively; lateral view, dorsal to the right. Arrowheads point at expression in areas of the developing eye, hindbrain and neural crest; anterior to the top. a1–a6 Transverse sections of embryo in (a) at the respective levels; dorsal to the top. c Schematic diagram of atoh8 distribution in brain tissue. d Flat mount of a 16-hpf embryo showing atoh8 expression; dorsal view, anterior to the left. Flat mount (anterior to the left) of 16-hpf embryo double-stained for atoh8 and myoD. e, e1–e3 transverse sections (dorsal to the top) through levels indicated in e. f, gatoh8/crestin signal distribution; arrowheads indicating presence/absence of atoh8 transcripts (violet) and crestin transcripts (red). h Whole embryo showing crestin expression in the head region; dorsal view, anterior to the left. Scale bars: whole embryos, flat mounts 200 µm; transverse sections 100 µm

a–c Whole embryos at 18 hpf showing atoh8, atoh8/myoD and crestin expression, respectively; lateral view, anterior to the top. Arrowhead in a marks the chordoneural hinge. d Schematic overview showing distribution of cell and tissue types from anterior (top) to posterior positions in the embryo (d1–d5) and in the whole embryo viewed from laterally (d6). Scale bar 200 µm

atoh8, atoh8/myoD and atoh8/crestin expression in whole embryos at 19–20 hpf (lateral view, dorsal to the right) and transverse sections of a, b and c at levels indicated. Green arrowheads in a1–a6 indicate the position of neural crest cells. Scale bars: whole embryos 200 µm, transverse sections 100 µm

atoh8, atoh8/myoD and atoh8/crestin expression profile at 22 hpf. a–c Whole mounts (lateral view, dorsal to the right) and associated serial transverse sections. d, d’ Flat mounts of zebrafish embryo showing expression in hindbrain, cerebellum and eye (dorsal view, anterior to the left). e Longitudinal sections of the hindbrain and cerebellum reveal the position of atoh8 signal in the superficial layer of the developing neural tissue; atoh8-positive streams of cells can be detected adjacent to the otic vesicle (black arrowhead); there is no clarity about the tissue type of these cells, since they seem to reside in areas apparently marked by crestin transcription (green arrowheads). f Transverse section through the diencephalon demonstrating atoh8 in the lens primordium and part of the retina (upper panel), and crestin at the temporal margin of the developing eye (lower panel). Scale bars: whole embryos 200 µm, flat mounts 100 µm, transverse sections 100 µm

atoh8, atoh8/myoD and atoh8/crestin expression at 24 hpf. a–c Whole mounts (lateral view, dorsal to the right) and a’, b’, c’ associated serial transverse sections. a1, a2 Flat mounts of zebrafish trunk; lateral view, anterior to the left. atoh8 is located in the deeper tissue of the somite, whereas myoD resides in the more superficial layers. Arrowheads in a, b and c pointing to hindbrain domains with strong (black arrowhead) and diminished (orange arrowhead) atoh8 signal. Arrowheads in (c’) pointing to neural crest cells of the lateral (light green) and medial (dark green) migratory pathway. Scale bars: whole embryos 200 µm, flat mounts 200 µm, transverse sections 100 µm